U.S. patent application number 13/007325 was filed with the patent office on 2011-07-21 for bioprocess and microbe engineering for total carbon utilization in biofuel production.
This patent application is currently assigned to Massachusetts Institute of Technology. Invention is credited to Gregory Stephanopoulos.
Application Number | 20110177564 13/007325 |
Document ID | / |
Family ID | 44277853 |
Filed Date | 2011-07-21 |
United States Patent
Application |
20110177564 |
Kind Code |
A1 |
Stephanopoulos; Gregory |
July 21, 2011 |
BIOPROCESS AND MICROBE ENGINEERING FOR TOTAL CARBON UTILIZATION IN
BIOFUEL PRODUCTION
Abstract
Some aspects of this invention provide methods and bioreactors
for converting a carbon source into a lipid. In some embodiments,
lipid production is carried out in an aerobic fermentor and carbon
dioxide generated during lipid production is converted into a
carbon substrate by CO.sub.2 fixation in an anaerobic fermentor. In
some embodiments, the carbon substrate generated by CO2 fixation is
used as the carbon source for lipid production, thus achieving
total carbon utilization in lipid production.
Inventors: |
Stephanopoulos; Gregory;
(Winchester, MA) |
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
|
Family ID: |
44277853 |
Appl. No.: |
13/007325 |
Filed: |
January 14, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61295302 |
Jan 15, 2010 |
|
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Current U.S.
Class: |
435/101 ;
435/134; 435/162; 435/170; 435/171; 435/289.1; 435/41 |
Current CPC
Class: |
C12P 7/6463 20130101;
C12P 7/10 20130101; Y02E 50/16 20130101; Y02E 50/30 20130101; C12P
7/16 20130101; Y02E 50/343 20130101; C12P 7/649 20130101; C12P 7/54
20130101; Y02E 50/13 20130101; C12M 23/58 20130101; C12M 43/00
20130101; Y02E 50/10 20130101 |
Class at
Publication: |
435/101 ; 435/41;
435/134; 435/162; 435/171; 435/170; 435/289.1 |
International
Class: |
C12P 19/04 20060101
C12P019/04; C12P 1/00 20060101 C12P001/00; C12P 7/64 20060101
C12P007/64; C12P 7/14 20060101 C12P007/14; C12P 1/02 20060101
C12P001/02; C12P 1/04 20060101 C12P001/04; C12M 1/00 20060101
C12M001/00; C12M 1/04 20060101 C12M001/04 |
Claims
1. A method comprising (a) culturing a first organism in the
presence of a carbon source under conditions suitable for the
organism to oxidize the carbon source, wherein the organism
produces CO.sub.2 as part of the oxidation process; and (b)
culturing a second organism in the presence of CO.sub.2 produced in
(a) under conditions suitable for the second organism to reduce the
CO.sub.2, wherein the organism produces a carbon substrate as part
of the reduction process.
2. The method of claim 1, wherein the conditions of (a) are
oxidizing conditions; the conditions of (a) are aerobic conditions;
the conditions of (b) are reducing conditions; and/or the
conditions of (b) are anaerobic conditions.
3.-5. (canceled)
6. The method of claim 1, wherein the culturing of (a) and/or (b)
is carried out in a fermentor.
7. The method of claim 1, wherein the culturing of (a) and of (b)
is carried out in separate fermentors; optionally, wherein the
culturing of (a) is carried out in an aerobic fermentor; and/or
optionally, wherein the culturing of (b) is carried out in an
anaerobic fermentor.
8.-9. (canceled)
10. The method of claim 1, further comprising contacting the first
organism with an oxidizing agent, optionally, wherein the oxidizing
agent is O.sub.2; and/or contacting the second organism with a
reducing agent, optionally, wherein the oxidizing agent is H.sub.2,
CO, syngas, or H.sub.2S.
11.-13. (canceled)
14. The method of claim 10, wherein the O.sub.2 and/or the H.sub.2
are generated by electrolysis of H.sub.2O.
15. (canceled)
16. The method of claim 7, wherein the culturing of (a) and/or (b)
is carried out in a liquid medium; optionally, wherein O.sub.2 is
dispersed in the liquid medium of the aerobic fermentor in the form
of micro-bubbles; and/or optionally, wherein H.sub.2 is dispersed
in the liquid medium of the anaerobic fermentor in the form of
micro-bubbles.
17. The method of claim 1, further comprising providing electrons
to the organism of (b) by contacting the organism of (b) with an
electric current.
18. (canceled)
19. The method of claim 1, wherein the carbon source is a
carbohydrate; optionally, wherein the carbohydrate is glucose,
fructose, ethanol, butanol, acetic acid, biomass, cellulose, or
hemicellulose.
20. (canceled)
21. The method of claim 1, wherein a product of the carbon source
oxidization process in (a) is a biofuels; a lipid; an edible lipid,
or a precursor thereof; the carbon substrate produced in (b) is a
biofuel; ethanol; biomass; cellulose; or hemi-cellulose; the carbon
substrate produced in (b) is a carbon source that can be oxidized
by the organism of (a); and/or the carbon source of (a) comprises
at least part of the carbon substrate produced in (b).
22.-32. (canceled)
33. The method of claim 1, wherein the organism of (a) and/or (b)
is a microorganism.
34. The method of claim 1, wherein the organism of (a) is an
oleaginous yeast, optionally, wherein the organism of (a) is Y.
lipolytica; and/or the organism of (b) is a CO.sub.2-fixing
bacterium, optionally, wherein the organism of (b) is an acetogenic
bacterium; and/or the organism of (a) and/or (b) is genetically
modified, optionally, wherein the organism of (a) overexpresses an
SCD gene.
35.-40. (canceled)
41. A bioreactor comprising (a) an aerobic fermentor, the aerobic
fermentor comprising (i) a carbon source, (ii) an organism
oxidizing the carbon source and generating CO.sub.2; and (iii) an
outflow, through which the CO.sub.2 is removed from the fermentor;
(b) an anaerobic fermentor comprising (i) an organism reducing
CO.sub.2, and (ii) an inflow providing CO.sub.2 to the fermentor,
wherein the inflow is connected to the outflow of (a)(iii).
42. The bioreactor of claim 41, wherein the aerobic and/or the
anaerobic fermentor comprises a liquid medium; the aerobic
fermentor comprises an oxidizing agent, optionally, wherein the
oxidizing agent is O.sub.f and/or the anaerobic fermentor comprises
a reducing agent, optionally, wherein the reducing agent is
H.sub.2, CO, syngas, or H.sub.2S.
43.-44. (canceled)
45. The bioreactor of claim 41, further comprising an electrolysis
apparatus that generates O2 and H2 from H2O, wherein the O2 is
delivered to the aerobic fermentor and/or the H2 is delivered to
the anaerobic fermentor.
46. The bioreactor of claim 41, wherein the aerobic fermentor
comprises O2 in the form of microbubbles and/or wherein the
anaerobic fermentor comprises H2 in the form of micro-bubbles,
and/or wherein the anaerobic fermentor comprises one or more
electrodes delivering an electric current to the fermentor in an
amount sufficient to provide the organism in the anaerobic
fermentor with electrons for CO.sub.2.
47. (canceled)
48. The bioreactor of any of claim 41, wherein the carbon source is
glucose, fructose, ethanol, butanol, acetic acid, biomass,
cellulose, or hemicellulose; a product of oxidizing the carbon
source is a biofuel; and/or a product of oxidizing the carbon
source is a lipid.
49.-51. (canceled)
52. The bioreactor of claim 41, wherein the aerobic fermentor
further comprises an outflow through which the product of oxidizing
the carbon source is removed.
53. The bioreactor of claim 41, wherein the inflow of (b)(ii) is
further connected to an external source of CO.sub.2.
54. The bioreactor of claim 41, wherein a product of CO.sub.2
reduction in the anaerobic fermentor is a carbon source that can be
oxidized by the organism in the aerobic fermentor; a product of
CO.sub.2 reduction in the anaerobic fermentor is biomass,
cellulose, or hemi-cellulose; a product of CO.sub.2 reduction in
the anaerobic fermentor is a biofuel; and/or a product of CO.sub.2
reduction in the anaerobic fermentor is ethanol or butanol.
55.-57. (canceled)
58. The bioreactor of claim 41, wherein the anaerobic fermentor
comprises an outflow through which a product of CO.sub.2 reduction
is removed; optionally, wherein the outflow through which the
product of CO.sub.2 reduction is removed from the anaerobic
fermentor is connected to the aerobic fermentor and the product of
CO.sub.2 reduction in the anaerobic fermentor is delivered to the
aerobic fermentor; optionally, wherein the product of CO.sub.2
reduction in the anaerobic fermentor constitutes at least part of
the carbon source in the aerobic fermentor.
59.-62. (canceled)
63. The bioreactor of claim 41, wherein the organism of (a) and/or
(b) is a microorganism; optionally, wherein the organism of (a) is
an oleaginous yeast; optionally, wherein the organism of (a) is Y.
lipolytica; optionally, wherein the organism of (b) is a
CO.sub.2-fixing bacterium; optionally, wherein the organism of (b)
is an acetogenic bacterium; and/or the organism of (a) and/or (b)
is genetically modified; optionally, wherein the organism of (a)
overexpresses an SCD gene.
64.-71. (canceled)
Description
RELATED APPLICATION
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to United States provisional patent application, U.S.
Ser. No. 61/295,302, filed Jan. 15, 2010, the entire contents of
which are incorporated herein by reference.
BACKGROUND
[0002] The main limitation in biodiesel production is the
availability of oil feedstock that is both expensive and in short
supply. Furthermore, yields of oil from oil seeds and vegetable oil
feedstocks (in gallons of gasoline equivalent per hectare and year)
are very low, approximately 1/5- 1/10 of the corresponding yields
of carbohydrate energy crops (1). Hence, conversion of carbohydrate
feedstocks to lipids and oils has attracted considerable attention
in recent years.
[0003] Production of oil by microbes, also referred to as microbial
oil or Single Cell Oil (SCO), has primarily been targeted for human
consumption and mostly restricted to commercial production of
dietary supplements, such as edible poly-unsaturated fatty acids
(PUFA) (2). The cost-effective use of microbial oil for biofuel
applications, especially as feedstock in biodiesel production,
requires several considerations such as, optimizing oil quality and
oil properties, ability to assimilate various carbon sources, high
final oil concentrations and, most importantly, high yields,
defined as actual grams of oil produced per gram of carbohydrate
substrate consumed. The importance of yield as defining factor in
economical biodiesel production stems from the large contribution
of the feedstock cost to the total biofuel manufacturing cost
(upwards of 55% by most estimates). Many microbes are known to
produce oil such as bacteria, yeast and algae with sub-optimal oil
yields (3). The low oil yield (along with low volumetric
productivity) is a central reason that microbial-derived oil has
failed to break commercial scale.
[0004] Several attempts have been made to produce oil using
bacterial species and yeasts. Examples include the oleaginous
bacterium Rhodococcus opacus (4) that achieved a yield close to 10%
which is one third of the maximum yield possible making the process
uneconomical (5). Recently, E. coli was genetically engineered to
directly secrete free fatty acids (6) or fatty acid methyl esters
(FAME/biodiesel) directly in the extracellular medium (7), at oil
conversion yields between 4.8% and 13%, compared to a maximum
theoretical yield of 30-33%. Similarly, oleaginous yeasts, such as
Yarrowia lipolytica, are being genetically modified for bio-oil
production (8). Current oil production using oleaginous yeasts
stands at 4-6 grams of oil per liter (9), which is considered very
low for commercial application.
[0005] In notable contrast to the low land-based productivity of
vegetable oil mentioned above is the use of microalgae for oil
production by direct photosynthetic fixation of atmospheric carbon
dioxide. The broad distribution of algal cultivation along with
lack of competition for food-producing cropland makes algal-derived
oils a compelling proposition (10), assuming that a number of
fundamental limitations will be satisfactorily resolved first (11).
These include the possibility that algal cultures may be
out-competed in open systems due to their slow growth rate relative
to other species, and very low algal and oil concentrations, which
makes dewatering and biomass processing extremely expensive.
SUMMARY
[0006] The conversion of carbohydrates to carbon substrates useful
for biofuel production (e.g., ethanol or triacylglycerides) in
living cells is an inefficient process. For example, approximately
half of the glucose feedstock in an ethanol fermentation process is
lost as CO.sub.2. This is the result of carbon oxidation that is
necessary to generate the reducing equivalents required to lower
the oxygen content of an initial glucose molecule
(C.sub.6H.sub.12O.sub.6) to that of the more reduced product
ethanol (C.sub.2H.sub.6O). This picture does not differ
significantly in other types of biofuels (e.g., butanol, lipids and
oils) produced from sugars such as glucose or xylose, products
themselves of either starch or cellulosic/hemicellulosic biomass
hydrolysis.
[0007] If the emitted CO.sub.2 could be captured, for example, by
using hydrogen from a non-fossil fuel source, the amount of land
required to produce a given amount of biofuel would be reduced by
two thirds (1). While thermochemical processes were contemplated in
the above CO.sub.2 fixation concept, aspects of this invention
provide biological methods to achieve fixation of CO.sub.2, which
have a higher overall yield as they operate closer to equilibrium
and are consequently more efficient. The use of the biological
methods for CO.sub.2 fixation provided herein is useful to increase
dramatically the amount of liquid fuels that can be obtained from a
certain land area. Additionally, some of these methods provide an
excellent means for hydrogen storage.
[0008] Some aspects of this invention provide methods,
microorganisms, and bioreactors for the generation of TAG from a
carbon source, in which CO.sub.2 generated during TAG production
from the carbon source is used in a biological CO.sub.2 fixation
process yielding a carbon substrate. Some aspects of this invention
provide methods, microorganisms, and bioreactors for heterotrophic
triacylglycerol (TAG) production from a carbon substrate that is a
product of biological CO.sub.2 fixation, for example, by anaerobic,
CO.sub.2 fixing organisms that utilize as reductant either gaseous
hydrogen or electrons provided by a biocathode. Some aspects of
this invention provide methods, microorganisms, and bioreactors for
the aerobic generation of TAG from a carbon substrate generated by
anaerobic CO.sub.2 fixation, wherein CO.sub.2 generated during
conversion of the carbon substrate is used in the anaerobic
CO.sub.2 fixation.
[0009] Biological CO.sub.2 fixation requires the concerted action
of various dehydrogenases (among other pathway enzymes), which are
typically obligate anaerobic enzymes. TAG production, on the other
hand, is an energy intensive process requiring the oxidation of
substantial amounts of carbon for the production of the energy and
reducing equivalents embodied in the production of TAG, the most
energy dense compound in nature. This should be, optimally, an
aerobic process, as the required amounts of energy for oil
production would be prohibitively slow to produce under anaerobic
conditions by substrate-level phosphorylation. The above
conflicting requirements suggest that it would be highly unlikely
to achieve both CO.sub.2 reduction and TAG production in the same
cellular environment.
[0010] Some aspects of this invention provide a solution to this
problem in separating the aerobic and anaerobic functions into two
different bioreactors: one for the intensely aerobic production of
TAG (e.g., oil) and the other for the anaerobic reduction of
CO.sub.2 in the presence of hydrogen or electric current. In some
embodiments, an oleaginous microbe optimized for TAG production
from a specific carbon substrate (e.g., carbohydrate feedstock) is
employed in the methods and bioreactors provided herein for the
aerobic conversion of a carbon source to TAG. In some embodiments,
non-photosynthetic, anaerobic CO.sub.2 fixation is achieved through
the use of bacteria, for example, acetogenic bacteria. In some
embodiments, the bacteria are genetically modified, or
pathway-engineered. In some embodiments, the bacteria are
Clostridia. In some embodiments, CO.sub.2 fixation is achieved
through the use of Clostridia under anaerobic culture conditions.
In some embodiments, CO.sub.2 fixation is achieved through direct
electron transfer from the biocathode of a reverse microbial fuel
cell (MFC).
[0011] Some aspects of this invention provide a method comprising
(a) culturing a first organism in the presence of a carbon source
under conditions suitable for the organism to oxidize the carbon
source, wherein the organism produces CO.sub.2 as part of the
oxidation process; and (b) culturing a second organism in the
presence of CO.sub.2 produced in (a) under conditions suitable for
the second organism to reduce the CO.sub.2, wherein the organism
produces a carbon substrate as part of the reduction process. In
some embodiments, the conditions of (a) are oxidizing conditions.
In some embodiments, the conditions of (a) are aerobic conditions.
In some embodiments, the conditions of (b) are reducing conditions.
In some embodiments, the conditions of (b) are anaerobic
conditions. In some embodiments, the culturing of (a) and/or (b) is
carried out in a fermentor. In some embodiments, the culturing of
(a) and of (b) is carried out in separate fermentors. In some
embodiments, the culturing of (a) is carried out in an aerobic
fermentor. In some embodiments, the culturing of (b) is carried out
in an anaerobic fermentor. In some embodiments, the method further
comprises contacting the first organism with an oxidizing agent. In
some embodiments, the oxidizing agent is O.sub.2. In some
embodiments, the method further comprises contacting the second
organism with a reducing agent. In some embodiments, the reducing
agent is H.sub.2, CO, syngas, or H.sub.2S. In some embodiments, the
O.sub.2 and/or the H.sub.2 are generated by electrolysis of
H.sub.2O. In some embodiments, the syngas is generated from coal or
natural gas. In some embodiments, the culturing of (a) and/or (b)
is carried out in a liquid medium. In some embodiments, O.sub.2 is
dispersed in the liquid medium of the aerobic fermentor in the form
of micro-bubbles; and/or H.sub.2 is dispersed in the liquid medium
of the anaerobic fermentor in the form of micro-bubbles. In some
embodiments, the method further comprises providing electrons to
the organism of (b) by contacting the organism of (b) with an
electric current. In some embodiments, the electric current is
provided via one or more electrodes. In some embodiments, the
carbon source is a carbohydrate. In some embodiments, the
carbohydrate is glucose, fructose, ethanol, butanol, acetic acid,
biomass, cellulose, or hemicellulose. In some embodiments, a
product of the carbon source oxidization process in (a) is a
biofuel. In some embodiments, the product of the carbon source
oxidization process in (a) is a lipid. In some embodiments, the
product of the carbon source oxidation is an edible lipid, or a
precursor thereof. In some embodiments, the carbon substrate
produced in (b) is a biofuel. In some embodiments, the carbon
substrate produced in (b) is ethanol. In some embodiments, the
carbon substrate produced in (b) is a carbon source that can be
oxidized by the organism of (a). In some embodiments, the carbon
substrate produced in (b) is acetic acid or acetate, biomass,
cellulose, or hemi-cellulose. In some embodiments, the carbon
substrate produced in (b) is processed for use as a carbon source
in (a). In some embodiments, the processing comprises hydrolysis of
at least part of the carbon substrate. In some embodiments, the
carbon source of (a) comprises at least part of the carbon
substrate produced in (b). In some embodiments, the carbon source
of (a) comprises the carbon substrate produced in (b). In some
embodiments, the carbon source of (a) consists of the carbon
substrate produced in (b). In some embodiments, the organism of (a)
is a microorganism. In some embodiments, the organism of (b) is a
microorganism. In some embodiments, the organism of (a) is an
oleaginous yeast. In some embodiments, the organism of (a) is Y.
lipolytica. In some embodiments, the organism of (b) is a
CO.sub.2-fixing bacterium. In some embodiments, the organism of (b)
is an acetogenic bacterium. In some embodiments, the organism of
(b) is a Clostridium sp. bacterium. In some embodiments, the
organism of (b) is C. acetobutylicum, C. ljungdahlii, C.
carboxydivorans, or C. autoethanogenum, C. thermohydrosulfuricum,
C. thermocellum, or C. thermoanaerofacter ethanoliticus, or any
other CO.sub.2 fixing microorganism descried herein. In some
embodiments, the organism of (a) and/or (b) is genetically
modified. In some embodiments, the organism of (a) overexpresses an
SCD gene or comprises any genetic modification described herein for
TAG-producing organisms, for example, in Example 1. In some
embodiments, the organism of (b) comprises a genetic modification
that increases the activity of a Wood-Ljungdahl metabolic pathway
member in the organism, or any modification described for CO.sub.2
fixing organisms herein.
[0012] Some aspects of this invention provide a bioreactor
comprising (a) an aerobic fermentor comprising (i) a carbon source,
(ii) an organism oxidizing the carbon source and generating
CO.sub.2, and (iii) an outflow, through which the CO.sub.2 is
removed from the fermentor; and (b) an anaerobic fermentor
comprising (i) an organism reducing CO.sub.2, and (ii) an inflow
providing CO.sub.2 to the fermentor, wherein the inflow is
connected to the outflow of the aerobic fermentor in (a)(iii). In
some embodiments, the aerobic and/or the anaerobic fermentor
comprises a liquid medium. In some embodiments, the aerobic
fermentor comprises an oxidizing agent and/or the anaerobic
fermentor comprises a reducing agent. In some embodiments, the
oxidizing agent is O.sub.2 and/or the reducing agent is H.sub.2,
CO, syngas, or H.sub.2S. In some embodiments, the bioreactor
further comprises an electrolysis apparatus that generates O.sub.2
and H.sub.2 from H.sub.2O, wherein the O.sub.2 is delivered to the
aerobic fermentor and/or the H.sub.2 is delivered to the anaerobic
fermentor. In some embodiments, the aerobic fermentor comprises
O.sub.2 in the form of microbubbles and/or wherein the anaerobic
fermentor comprises H.sub.2 in the form of micro-bubbles. In some
embodiments, the anaerobic fermentor comprises one or more
electrodes delivering an electric current to the fermentor in an
amount sufficient to provide the organism in the anaerobic
fermentor with electrons for CO.sub.2. In some embodiments, the
carbon source is glucose, glucose, fructose, ethanol, butanol,
acetic acid or acetate, biomass, cellulose, or hemicellulose. In
some embodiments, a product of oxidizing the carbon source is a
biofuel. In some embodiments, a product of oxidizing the carbon
source is a lipid. In some embodiments, the lipid is an edible
lipid or a precursor thereof. In some embodiments, the lipid is a
triacylglyceride (TAG). In some embodiments, the aerobic fermentor
further comprises an outflow through which the product of oxidizing
the carbon source is removed. In some embodiments, the inflow of
(b)(ii) is further connected to an external source of CO.sub.2. In
some embodiments, a product of CO.sub.2 reduction in the anaerobic
fermentor is a carbon source that can be oxidized by the organism
in the aerobic fermentor. In some embodiments, a product of
CO.sub.2 reduction in the anaerobic fermentor is biomass,
cellulose, or hemi-cellulose. In some embodiments, a product of
CO.sub.2 reduction is a biofuel. In some embodiments, a product of
CO.sub.2 reduction is ethanol or butanol. In some embodiments, a
product of CO.sub.2 reduction is acetic acid or acetate. In some
embodiments, the anaerobic fermentor comprises an outflow through
which a product of CO.sub.2 reduction is removed. In some
embodiments, the outflow through which the product of CO.sub.2
reduction is removed from the anaerobic fermentor is connected to
the aerobic fermentor and the product of CO.sub.2 reduction in the
anaerobic fermentor is delivered to the aerobic fermentor. In some
embodiments, the product of CO.sub.2 reduction in the anaerobic
fermentor constitutes at least part of the carbon source in the
aerobic fermentor. In some embodiments, the product of CO.sub.2
reduction in the anaerobic fermentor constitutes the carbon source
in the aerobic fermentor.
[0013] In some embodiments, the influx of carbon into the
bioreactor is limited to the influx of CO.sub.2 into the anaerobic
fermentor. In some embodiments, the organism of (a) is a
microorganism. In some embodiments, the organism of (b) is a
microorganism. In some embodiments, the organism of (a) is an
oleaginous yeast. In some embodiments, the organism of (a) is Y.
lipolytica. In some embodiments, the organism of (b) is a
CO.sub.2-fixing bacterium. In some embodiments, the organism of (b)
is an acetogenic bacterium. In some embodiments, the organism of
(b) is a Clostridium sp. bacterium. In some embodiments, the
organism of (b) is C. acetobutylicum, C. ljungdahlii, C.
carboxydivorans, C. autoethanogenum, C. thermohydrosulfuricum, C.
thermocellum, or C. thermoanaerofacter ethanoliticus. In some
embodiments, the organism of (a) and/or (b) is genetically
modified. In some embodiments, the organism of (a) overexpresses an
SCD gene or comprises any genetic modification described herein for
TAG-producing organisms, for example, in Example 1. In some
embodiments, the organism of (b) comprises a genetic modification
that increases the activity of a Wood-Ljungdahl metabolic pathway
member in the organism, or any modification described for CO.sub.2
fixing organisms herein.
[0014] Other advantages, features, and uses of the invention will
be apparent from the detailed description of certain non-limiting
embodiments, the drawings, which are schematic and not intended to
be drawn to scale, and the claims.
BRIEF DESCRIPTION OF THE D RAWINGS
[0015] FIG. 1. The Wood-Ljungdahl pathway. "H.sub.2" is used in a
very general sense to designate the requirement for two electrons
and two protons in the reaction.
[0016] FIG. 2. Schemes of combining an aerobic oil producing
fermentation with an anaerobic CO.sub.2 fixing process.
[0017] FIG. 3. Oleaginous microbe and time course (hrs) of oil
accumulation in fermentor.
[0018] FIG. 4. Growth and oil production of oleaginous microbe on
acetate.
[0019] FIG. 5. Schematic of the 96-well MFC and experimental
set-up. A) Cross-section of bioreactor showing components,
including bottom plate electrode and flow path; B) Assembled
reactor. A Gamry single-channel potentiostat and 12-channel
multiplexer (not shown) are attached to the pins of the completed
reactor below.
[0020] FIG. 6. Growth and acetate production of Acetobacterium
woodii on fructose and H.sub.2/CO.sub.2. (71)
[0021] FIG. 7. Sample process flow diagram with basis of 500 kg/hr
oil production (approx. 1M gallons/yr).
DEFINITIONS
[0022] The term "acetogen" or "acetogenic microbe" or "acetogenic
bacterium" is art-recognized and refers to a microorganism that
generates acetate as a product of anaerobic CO.sub.2 fixation.
Acetogens are found in a variety of anaerobic habitats and can use
a variety of compounds as sources of energy and carbon; the best
studied form of acetogenic metabolism involves the use of carbon
dioxide as a carbon source and hydrogen as an energy source.
[0023] The term "aerobic conditions" is art recognized and refers
to conditions that provide sufficient oxygen for efficient
oxidation of a carbon source by an aerobic organism. In some
embodiments, aerobic conditions are conditions that provide an
abundance or even an overabundance of oxygen, for example, in the
form of micro-bubbles of oxygen in a liquid medium. For example, a
fermentor comprising a gaseous phase comprising at least 10%, at
least 15%, at least 20%, at least 30%, at least 50%, or more oxygen
is referred to as an aerobic fermentor.
[0024] The term "anaerobic conditions" is art recognized and refers
to conditions that do not provide sufficient oxygen for efficient
carbon oxidation by an aerobic organism. In some embodiments,
anaerobic conditions are characterized by the essential absence of
oxygen. In other embodiments, the oxygen content is less than
required by a microbe employed to efficiently oxidate a carbon
source. For example, a fermentor comprising a liquid medium and a
gaseous phase comprising less than 5%, less than 2%, less than 1%,
less than 0.5%, less than 0.1%, less than 0.01%, or less than
0.001% oxygen is referred to as an anaerobic fermentor.
[0025] The term "biofuel" refers to a fuel that is derived from a
biological source, such as a living cell, microbe, fungus, or
plant. The term includes fuel directly obtained from a biological
source, for example, by conventional extraction, distillation, or
refining methods, and fuel produced by processing a biofuel
precursor obtained from a biological source, for example by
chemical modification, such as transesterification procedures.
Examples of biofuels that are directly obtainable are alcohols such
as ethanol, propanol, and butanol, fat, and oil. Examples of
biofuels that are obtained by processing of a biofuel precursor
(e.g., a lipid, such as a TAG), are biodiesel (e.g., produced by
transesterification of a lipid), and green diesel/modified oil
fuels (e.g., produced by hydrogenation of an oil). Biodiesel, also
referred to as fatty acid methyl (or ethyl) ester, is one of the
economically most important biofuels today and can be produced on
an industrial scale by transesterification of lipids, in which
sodium hydroxide and methanol (or ethanol) reacts with a lipid, for
example, a triacylglycerol, to produce biodiesel and glycerol.
[0026] The term "biomass" refers to material produced by growth
and/or propagation of a living cell or organism, for example, a
microbe. Biomass may contain cells, microbes, plants, and/or
intracellular contents, for example cellular fatty acids and TAGs,
as well as extracellular material. Extracellular material includes,
but is not limited to, compounds secreted by a cell, for example,
secreted fatty acids or TAGs. In some embodiments, biomass is
processed before being used as a carbon source for aerobic biofuel
production. For example, biomass comprising a high content of
non-fermentable carbohydrates, such as cellulose, can be hydrolyzed
into fermentable carbohydrates by methods known to those of skill
in the art. In some embodiments, the pretreatment of biomass
feedstock includes depolymerizing cellulose and/or hemicellulose
components to monomeric sugars using a pretreatment method known to
those of skill in the art, for example, a dilute acid or ammonia
fiber expansion (AFEX) method (see, e.g., Yang B, Wyman C E. Dilute
acid and autohydrolysis pretreatment. Methods Mol Biol. 2009;
581:103-14; Balan V, Bals B, Chundawat S P, Marshall D, Dale B E,
Lignocellulosic biomass pretreatment using AFEX Methods Mol Biol.
2009; 581:61-77). Other methods for depolymerization of biomass
polymers to monomeric sugars are well known to those of skill in
the art and are contemplated to be used in some embodiments of this
invention.
[0027] The term "culturing" refers to maintaining a culture of an
organism, for example, a microbe described herein for a period of
time, generally, for a period of time sufficient for a desired
fermentation process to be carried out by the microbe. In some
embodiments, the culture comprises a microbe described herein and a
medium, for example, a liquid medium. In some embodiments, the
culture comprises a carbon source, for example a carbon source
dissolved in the culture medium. For example, in some embodiments,
a microbe is cultured in an aerobic fermentor in a liquid medium in
the presence of a carbon source (e.g., acetate, or a soluble sugar)
dissolved in the medium. In some embodiments, the culture comprises
a salt and/or buffer establishing conditions of salinity,
osmolarity, and pH, that are amenable to survival, growth, and/or
conversion of the carbon source to a biofuel or biofuel precursor
by the cultured organism. In some embodiments, the culture
comprises an additional component, for example, an additive.
Non-limiting examples of additives are nutrients, enzymes, amino
acids, albumin, growth factors, enzyme inhibitors (for example
protease inhibitors), fatty acids, lipids, hormones (e.g.,
dexamethasone and gibberellic acid), trace elements, inorganic
compounds (e.g., reducing agents, such as manganese),
redox-regulators (e.g., antioxidants), stabilizing agents (e.g.,
dimethylsulfoxide), polyethylene glycol, polyvinylpyrrolidone
(PVP), gelatin, antibiotics (e.g., Brefeldin A), salts (e.g.,
NaCl), chelating agents (e.g., EDTA, EGTA), and enzymes (e.g.,
cellulase, dispase, hyaluronidase, or DNase). In some embodiments,
the culture may comprise a compound, for example, a small molecule
compound or drug, inducing or inhibiting transcription from a
conditional or inducible promoter, for example doxicycline,
tetracycline, tamoxifen, IPTG, hormones, or metal ions. While the
specific culture conditions, for example, the concentration of the
carbon source, will depend upon the respective engineered
microorganism to be cultured, general methods and culture
conditions for the generation of microbial cultures are well known
to those of skill in the art, and are described, for example, in J.
Sambrook and D. Russell, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press; 3rd edition (Jan. 15, 2001);
David C. Amberg, Daniel J. Burke; and Jeffrey N. Strathern, Methods
in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual,
Cold Spring Harbor Laboratory Press (April 2005); John N. Abelson,
Melvin I. Simon, Christine Guthrie, and Gerald R. Fink, Guide to
Yeast Genetics and Molecular Biology, Part A, Volume 194 (Methods
in Enzymology Series, 194), Academic Press (Mar. 11, 2004);
Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and
Molecular and Cell Biology, Part B, Volume 350 (Methods in
Enzymology, Vol 350), Academic Press; 1st edition (Jul. 2, 2002);
and Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics
and Molecular and Cell Biology, Part C, Volume 351, Academic Press;
1st edition (Jul. 9, 2002), all of which are incorporated by
reference herein.
[0028] The term "fermentor" refers to an enclosure, or partial
enclosure, in which a biological and/or chemical reaction takes
place, at least part of which involves a living organism or part of
a living organism. Where liquid cultures are used for fermentation,
the fermentor is typically a culture vessel able to hold the
desired amount of liquid media. If a gaseous phase is employed in
the fermentation process, the fermentor employed will have a volume
allowing accommodation of the gaseous phase and, if the gaseous
phase is not air, the fermentor is typically sealed in an airtight
manner. Typically, a fermentor comprises one or more inflows and/or
outflows for the introduction and/or removal of liquids, solids,
and/or gas into and/or out of the fermentor. Suitable fermentor
configurations will be apparent to those of skill in the art. For
example, in some embodiments, a continuous stirred tank reactor
(CTSR), a bubble column reactor (BCR) or a trickle bed reactor
(TBR), may be employed. In some embodiments, a fermentor comprises
a culture of microbes performing the fermentation process. In some
embodiments, a fermentor may continuously or semi-continuously be
fed with new microbes from a growth or culture vessel. Depending on
the fermentation scale, fermentors can range from volumes of
milliliters to thousands of liters or more. Some fermentors
according to aspects of this invention may include cell cultures
where microbes are in contact with moving liquids and/or gas
bubbles. Microbes or microbe cultures in accordance with aspects of
this invention may be grown in suspension or attached to solid
phase carriers. Non-limiting examples of carrier systems include
microcarriers (e.g., polymer spheres, microbeads, and microdisks
that can be porous or non-porous), cross-linked beads (e.g.,
dextran) charged with specific chemical groups (e.g., tertiary
amine groups), 2D microcarriers including cells trapped in
nonporous polymer fibers, 3D carriers (e.g., carrier fibers, hollow
fibers, multicartridge reactors, and semi-permeable membranes that
can comprising porous fibers), microcarriers having reduced ion
exchange capacity, encapsulation cells, capillaries, and
aggregates. Carriers can be fabricated from materials such as
dextran, gelatin, glass, and cellulose.
[0029] The term "lipid" refers to fatty acids and their
derivatives. Accordingly, examples of lipids include fatty acids
(FA, both saturated and unsaturated); glycerides or glycerolipids,
also referred to as acylglycerols (such as monoglycerides
(monoacylgycerols), diglycerides (diacylglycerols), triglycerides
(triacylglycerols, TAGs, or neutral fats); phosphoglycerides
(glycerophospholipids); nonglycerides (sphingolipids, sterol
lipids, including cholesterol and steroid hormones, prenol lipids
including terpenoids, fatty alcohols, waxes, and polyketides); and
complex lipid derivatives (sugar-linked lipids or glycolipids, and
protein-linked lipids). Lipids are an essential part of the plasma
membrane of living cells and microbes. Some cells and microbes also
produce lipids to store energy, for example in the form of
triacylglycerols in lipid droplets.
[0030] The term "syngas" is art recognized and refers to a gas
mixture that contains varying amounts of carbon monoxide and
hydrogen, and frequently also carbon dioxide. Syngas can be
produced from coal, first by pyrolysis to coke (destructive
distillation), followed by alternating blasts of steam and air, or
from biomass or municipal waste. Syngas can also be produced by
steam reforming of natural gas or liquid hydrocarbons. In some
embodiments, syngas is introduced into the anaerobic fermentor to
provide reductants (CO and H.sub.2) and, in some cases, also
CO.sub.2 for fixation by a CO.sub.2 fixing organism. In some
embodiments, natural gas or coal is used to produce syngas, which
is then used according to aspects of this invention to produce a
lipid, for example a TAG. In some embodiments, the TAG is a
biofuel. In some embodiments, the TAG is an edible lipid or a
precursor of an edible lipid. Accordingly, some aspects of this
invention provide methods to convert an inorganic carbon source
(e.g., coal or natural gas) into a biofuel or an edible lipid, for
example, via syngas distillation and biological fermentation steps
as described herein
[0031] The term "triacylglyceride" (TAG, sometimes also referred to
as triacylglycerol or triglyceride) refers to a molecule comprising
a single molecule of glycerol covalently bound to three fatty acid
molecules, aliphatic monocarboxylic acids, via ester bonds, one on
each of the glycerol molecule's three hydroxyl (OH) groups.
Triacylglycerols are highly concentrated stores of metabolic energy
because of their reduced, anhydrous nature, and are a suitable
feedstock for biodiesel production.
DETAILED DESCRIPTION
[0032] Some aspects of this invention provide novel bioprocessing
methods, microorganisms, and bioreactors for the production of
lipids, for example, of TAGs. Some aspects of this invention
provide methods and bioreactors in which CO.sub.2 generated during
the aerobic conversion of a carbon source to lipid (e.g., TAG), for
example, in an aerobic fermentor, is used in an anaerobic CO.sub.2
fixation process yielding a carbon substrate, for example, in a
separate, anaerobic fermentor. In some embodiments, the carbon
substrate produced by anaerobic CO.sub.2 fixation is itself a
biofuel, for example, ethanol. In some embodiments, the carbon
substrate produced by anaerobic CO.sub.2 fixation is a compound
that can be used as the carbon source for the aerobic production of
lipid (e.g., TAG), for example, acetate. In some embodiments,
methods and bioreactors are provided for the production of biofuel
(e.g. TAG and/or TAG precursors or derivatives, such as fatty acids
or biodiesel) from CO.sub.2 and H.sub.2 or electrons provided by
electric current.
[0033] In some embodiments, a TAG-producing microorganism capable
of converting a carbon substrate, e.g., a carbohydrate feedstock or
an organic compound (e.g., acetate), to a TAG that can be used for
biodiesel (e.g., fatty acid methyl ester, FAME) production or the
production of edible lipids or other TAGs or TAG derivatives as
described herein, is employed for TAG production in an aerobic
fermentor. In some embodiments, the microorganism is an oleaginous
microorganism, for example, an oleaginous yeast. In some
embodiments, the oleaginous yeast employed is Yarrowia lipolytica.
In some embodiments, aerobic TAG fermentation is combined with
anaerobic CO.sub.2 fixing bacteria operating in a separate
anaerobic fermentor. In some embodiments, electrons are provided to
the anaerobic fermentor via hydrogen for reducing potential. In
some embodiments, electrons are provided to the anaerobic fermentor
via current (e.g., via electrodes) for reducing potential. In some
embodiments, the product of the anaerobic CO.sub.2 fixation is a
biofuel, for example, an alcohol, such as ethanol. In some
embodiments, the product of the anaerobic CO.sub.2 fixation is a
carbon substrate, e.g., acetate, that can be used for aerobic
fermentation to TAG. In some embodiments, acetate produced by
anaerobic CO.sub.2 fixation is utilized by the aerobic
microorganism for growth and TAG (e.g., oil) production. In some
embodiments, the aerobic acetate-to-TAG conversion achieves
close-to-theoretical yields.
[0034] The economic viability of some of the methods provided
herein depends on the rate of CO.sub.2 fixation and acetate
production. While typical reported volumetric rates are low,
specific rates of CO.sub.2 fixation by acetogens are reasonable and
can be significantly enhanced by applying technologies of metabolic
engineering and synthetic biology. Some aspects of this invention
provide methods for the engineering and/or isolation of organisms
capable of rapid fixation of CO.sub.2 and acetate production. In
some embodiments, enhanced
[0035] CO.sub.2 fixation is accomplished by engineering the
CO.sub.2 fixation pathway in a CO.sub.2 fixing microorganism, e.g.,
an acetogenic bacterium, in order to amplify carbon flux. In some
embodiments, a natural organism or a mutant derivative of a natural
organism that can efficiently accept electrons for CO.sub.2
reduction in a reverse microbial fuel cell configuration is
isolated and used for CO.sub.2 fixation.
Anaerobic CO.sub.2 Fixation Using Microbes
[0036] In some embodiments, CO.sub.2 is converted to a carbon
substrate by a microorganism via anaerobic CO.sub.2 fixation. In
some embodiments, the carbon substrate is a biofuel, for example,
an alcohol, such as ethanol. In some embodiments, the carbon
substrate is a compound that can be used as a carbon source for
aerobic fermentation to a TAG, for example, acetate.
[0037] Naturally occurring acetogens (acetogenic bacteria) that can
produce acetate by fixing CO.sub.2 in the presence of hydrogen or
other electron source are well known to those of skill in the art
and include, but are not limited to acetogenic Clostridia. While
the overall rates of acetate production are low, the specific rates
are reasonable, such that, if one could achieve a dense culture of
approximately OD35-50, while maintaining the same specific rates of
CO.sub.2 fixation and acetate production, the overall acetate
productivity could approach that of ethanol production by yeast.
This would make the envisioned process of biodiesel production from
CO.sub.2 fixation economically feasible at a maximum hydrogen price
in the range of $1.50-1.70. In some embodiments, acetogenic
microbes are provided that are further enhanced to exhibit
increased specific metabolic rates via methods of metabolic
engineering and synthetic biology.
[0038] Suitable organisms and culture/fermentation conditions for
conversion of CO.sub.2 to a carbon substrate, for example, acetic
acid, butanol, or ethanol are described herein and additional
suitable organisms and culture/fermentation conditions are well
known to those of skill in the art and include, but are not limited
to the organisms and culture or fermentation conditions described
in International Patent Application Publication Nos: WO2009/105372;
WO2007/117157; WO2008/115080; and WO2009/064200; the entire
contents of each of which are incorporated herein by reference.
Additional suitable organisms and culture/fermentation conditions
include, but are not limited to, those described in Das, A. and L.
G. Ljungdahl, Electron Transport System in Acetogens; Drake, H. L.
and K. Kusel, Diverse Physiologic Potential of Acetogens; Chapters
14 and 13 of Biochemistry and Physiology of Anaerobic Bacteria, L.
G. Ljungdahl eds,. Springer (2003); U.S. Patent Application
Publication No. 2007/0275447 entitled "Indirect Or Direct
Fermentation of Biomass to Fuel Alcohol," (e.g., Clostridium
carboxidivorans); and U.S. Pat. No. 7,704,723 entitled "Isolation
and Characterization of Novel Clostridial Species," (e.g.,
Clostridium ragsdalei); the entire contents of each of which are
incorporated herein by reference. Additional suitable
microorganisms include, but are not limited to, Butyribacterium
methylotrophicum (see, e.g., "Evidence for Production of n-Butanol
from Carbon Monoxide by Butyribacterium methylotrophicum," Journal
of Fermentation and Bioengineering, vol. 72, 1991, p. 58-60;
"Production of butanol and ethanol from synthesis gas via
fermentation," FUEL, vol. 70, May 1991, p. 615-619); Clostridium
Ljungdahli, (see, e.g., U.S. Pat. Nos. 6,136,577; 5,173,429,
5,593,886, and 6,368,819; International Patent Application
Publication Nos WO 00/68407; WO 98/00558 and WO 02/08438; and
European Patent EP 117309); Clostridium autoethanogenum (see, e.g.,
Aribini et al, Archives of Microbiology 161: pp 345-351); Moorella
sp. (see, e.g., Sakai et al, Biotechnology Letters 29: pp
1607-1612). The entire contents of each of the above listed
publications is incorporated herein by reference. In some
embodiments, the acetogenic bacterium is a Clostridium, Moorella,
Oxobacter, Peptostreptococcus, Acetobacterium, Eubacterium,
Desulfotomaculum, Archaeglobulus or Butyribacterium, for example,
Clostridium carboxidivorans, Butyribacterium methylotrophicum,
Clostridium tetanomorphum, Clostridium autoethanogenum, Clostridium
ljungdahlii, Clostridium carboxidivorans, Clostridium
tetanomorphum, Oxobacter pfennigii, Peptostreptococcus productus,
Acetobacterium woodii, Eubacterium limosum, Butyribacterium
methylotrophicum, Moorella thermoacetica, Moorella
thermoautotrophica, Desulfotomaculum kuznetsovii, Desulfotomaculum
thermobenzoicum, or Archaeoglobulus fulgidis. Additional acetogens
suitable for use in the methods and anaerobic fermentors disclosed
herein will be apparent to those of skill in the art. It will also
be appreciated that, while in preferred embodiments a homogeneous
culture of acetogens of a single strain is employed, a mixed
culture of two or more acetogens may also be used in a CO.sub.2
fixation process or fermentor as provided herein.
[0039] In some embodiments, the CO.sub.2 fixing bacteria (e.g., the
acetogen) is cultured in the anaerobic fermentor in a suitable
liquid medium. In some embodiments, the liquid medium comprises
vitamins and minerals sufficient to permit growth of the
microorganism used. Suitable liquid media for anaerobic microbe
culture are known to those of skill in the art and include, but are
not limited to, those described in U.S. Pat. Nos. 5,173,429; and
5,593,886; and International Patent Application Publication No WO
02/08438; the entire contents of each of which are incorporated
herein by reference.
Acetogenic Bacteria, Clostridia and Non-Photosynthetic CO.sub.2
Fixation
[0040] Many of the non-photosynthetic carbon fixation pathways
belong to anaerobic metabolism. These pathways have been
hypothesized to be similar to the primordial activity of life
billions of years ago, where inorganic compounds were plentiful and
organism complexity was very low. Three of them are the (1)
Wood-Ljungdahl (W-L) pathway, (2) reductive TCA Cycle, and (3)
3-Hydroxypropionate pathway (12), of which the most prominent is
the Wood-Ljungdahl pathway. While other pathways for carbon
fixation are cyclic, requiring the recycling of intermediates, the
Wood-Ljungdahl pathway is the only linear pathway known to fix
carbon. It is further suggested that this pathway may have been the
first autotrophic process on Earth (13).
[0041] Besides using the Wood-Ljungdahl pathway to grow using
CO.sub.2 as a sole carbon source, some bacteria (e.g. acetogens)
employ this pathway in order to maximize yield when grown with
other substrates (e.g., glucose). They do this, for example, by
consuming glucose normally by glycolysis, which produces CO.sub.2,
reducing equivalents (e.g., NADH), and carbon for biomass or
product (e.g., pyruvate). In certain circumstances, the cell can
recover the CO.sub.2 by using those same reducing equivalents to
form acetyl-CoA (14). This allows acetogens to exhibit a maximum
theoretical yield of 100%. All glucose consumed is metabolized to
acetate, rather than the 50-60% as observed in many other organisms
(15).
[0042] While most organisms employing glycolysis can only produce a
net of 2 ATP for every mole of glucose, acetogens are able to
produce 4 ATP. However, when using CO.sub.2 as the sole carbon
source, no net ATP is generated by substrate-level phosphorylation
as shown in FIG. 1 and Eq (1). This process produces very little
energy, which is captured by ion gradients--probably suggesting
these organisms grow rather slowly. Energy stored in ion gradients
is converted to ATP by an F.sub.1F.sub.0 ATP synthase (16). The
overall reaction for the fixation of carbon using this pathway is
presented in equation (1). This process is referred to as
homo-acetogenesis, or the acetyl-CoA Wood-Ljungdahl pathway.
2CO.sub.2+4H.sub.2.fwdarw.CH.sub.3COOH+H.sub.2O (Eq. 1)
[0043] Each CO.sub.2 molecule proceeds down a separate branch of
this pathway. One CO.sub.2 is activated by an equivalent of ATP,
proceeds down the `methyl (or Eastern) branch`, and is reduced to
an activated methyl group while the other proceeds down the
`carbonyl (or Western) branch` and is reduced to CO. At this point
the two separate branches merge and one molecule of acetyl-CoA is
synthesized. This acetyl-COA may then undergo substrate-level
phosphorylation, thereby being reduced to acetate and regenerating
the ATP used to activate the CO.sub.2 molecule that entered the
`methyl branch`.
[0044] The `methyl branch` of the acetyl-CoA Wood-Ljungdahl pathway
depends on six different enzymes: formate dehydrogenase,
formyl-H.sub.4F synthetase, methenyl-H.sub.4F cyclohydrolase,
methylene-H.sub.4F dehydrogenase, methylene-H.sub.4F reductase, and
methyltransferase (13). However, the first five of these genes are
found in nearly all bacteria and eukaryotes (17). Conversely, the
`carbonyl branch` of the acetyl-CoA Wood-Ljungdahl pathway depends
only on the single gene acetyl-CoA synthase (13) and this gene is
unique to acetogens, methanogens, and sulfate reducers.
Furthermore, the acetyl-CoA synthase enzyme is bifunctional. Not
only does it catalyze the reduction to CO, but also the assembly of
the carbon from both branches into acetyl-CoA (17). Transformation
of this pathway into any model bacteria or yeast, therefore, will
only require the heterologous expression of two genes:
methyltransferase and acetyl-CoA synthase. In some embodiments, any
acetogenic microbe modified to exhibit heterologous expression of
methyltransferase and/or acetyl-CoA synthase is suitable for use in
the methods and/or fermentors provided herein.
[0045] The W-L pathway exists in several organisms, best known
among them being the acetogenic Clostridia, such as Clostridium
aceticum, Clostridium difficile, Moorella thermoacetica (formerly
Clostridium) and, also, Acetobacterium woodii. Besides a strong
medical interest in these organisms, it is also noted that
Clostridia have been the organism of choice for the biological
production of solvents and butanol (18-19). As a result, there is a
large body of research on their growth and physiology, enzymology
of many reactions, including those listed above associated with the
W-L pathway as well as enzymes catalyzing the
Acetone-Butanol-Ethanol (ABE) pathway (20-21). In addition, the
genomes of two solventogenic Clostridia have been sequenced and
numerous studies have examined their transformation with homologous
and heterologous genes. Metabolic Engineering of solventogenic
Clostridia has been advanced in the past 20 years mostly by the
work of the Papoutsakis laboratory, which developed vectors,
promoters, transformation systems, and numerous strains with
varying but well-defined genetic backgrounds in their studies of
gene expression, pathway construction and product formation
(21-23).
[0046] Some aspects of this invention are based on the recognition
that deployment of Clostridia for CO.sub.2 fixation at industrial
scale can be achieved by: (a) increasing the capacity of the
CO.sub.2-fixing pathway by over-expressing properly identified and
targeted genes in order to enhance the specific CO.sub.2
assimilation rates, and/or, (b) growing CO.sub.2-fixing Clostridia
to high cell densities in order to achieve high volumetric
productivities. Some methods and engineered microbes provided
herein achieve these goals, either alone or in combination.
Metabolic Engineering of Clostridia
[0047] In some embodiments, a natural acetogen (preferably
Clostridium aceticum, Clostridium thermoaceticum, Moorella
thermoacetica), is used as the CO.sub.2 fixing organism in the
anaerobic fermentor. In other embodiments, engineered acetogen
strains, for example, of C. acetobutylicum, as described elsewhere
herein, are employed as the CO.sub.2 fixing organism in the
anaerobic fermentor.
[0048] Some aspects of this invention relate to the improvement of
the overall volumetric productivity of acetate production via
metabolic engineering of acetogens. Some embodiments provide
methods for increasing the acetogen culture density as well as
methods for engineering the pathway of acetogenesis to enhance the
specific rate of hydrogen adsorption and metabolic processing.
[0049] Two seminal advances in the molecular biology of Clostridia
should be noted. The first is the collective work on engineering
the solventogenic pathways that resulted in several very
interesting engineered strains. Using a combination of gene (ptb or
solR) knockouts with plasmid-borne expression of the
aldehyde-alcohol Dehydrogenase (DH) gene aad, generated strains
capable of the highest reported solvent (butanol/acetone/ethanol)
production in this or any other organism (49-50). More directly
related to some aspects of the instant invention, the issue of
aerotolerance has been recently attended to (51). It was shown that
deletion of a peroxide repressor (PerR)-homologous protein in C.
acetobutylicum resulted in prolonged aerotolerance, limited growth
under aerobic conditions, higher resistance to H.sub.2O.sub.2, and
rapid consumption of oxygen. This has practical implications in
allowing the cells to carry out partial aerobic metabolism for
increasing cell densities and resolving electron flow
bottlenecks.
[0050] Another major advance in the field of clostridial
biotechnology is the abolition of sporulation while maintaining
solvent formation. This was accomplished by disrupting the genes
coding for two major sporulation-specific sigma factors (SigE and
SigG) (52). In a different approach, asporogenous,
non-solventogenic strains (such as strains M5 and DG1 of C.
acetobutylicum, which have lost the pSOL1 megaplasmid (53)) were
used as a starting point and the desirable solvent formation genes
were re-introduced or overexpressed. This resulted in high yielding
strains whose product distribution could be additionally controlled
(22).
[0051] In some embodiments, an acetogenic strain of clostridia is
employed for anaerobic CO2 fixation that exhibits one or more
modifications as described herein, for example, a peroxide
repressor deletion, which allows growth at increased densities.
Metabolic Engineering for Enhancing Acetate Formation in C.
acetobutylicum
[0052] In some embodiments, a metabolically engineered C.
acetobutylicum is provided that directly utilizes CO.sub.2 and
H.sub.2 for the production of acetate and biomass. In some
embodiments, genes from the Wood-Ljungdahl pathway are isolated
from other mesophilic or thermophilic clostridia and cloned into C.
acetobutylicum. C. acetobutylicum does not have a complete, native
Wood-Ljungdahl pathway but does have a number of homologs to
components of the W-L pathway (Table 1).
TABLE-US-00001 TABLE 1 Homologs of the W-L pathway in Moorella
thermoacetica, C. difficile and C. acetobutylicum. Genes of
Woods-Ljungdahl pathway in: M. thermo- C. aceto-butylicum C.
difficile Protein identity between: acetica (MTA) (CAC) (CDF)
MTA/CAC MTA/CDF CDF/CAC Eastern Moth_2312 -- CD3317 -- 32.7% --
Moth_2314 CAC0764 CD1537 21.5% 26.6% 37.1% Moth_0109 CAC3201 CD0718
64.8% 66.2% 62.1% Moth_1516 CAC2083 CD0720 44.1% 42.1% 37.1%
Moth_1191 CAC0291 CD0722 12.5% 38.6% 13.1% Western Moth_1197
CAC0578 CD0727 6.3% 37.6% 6.8% Moth_1201 -- CD0726 -- 37.8% --
Moth_1198 -- CD0725 -- 38.0% -- Moth_1203 CAC2498/0116 CD0716
29.8%/27.5% 38.5% 30.2%/29.8% Moth_1202 -- CD0728 -- 46.1% --
[0053] The first homolog is the .beta.-subunit of the formate
dehydrogenase, which reduces CO.sub.2 to formate and is the first
reaction in the "Eastern" branch of the pathway. In M.
thermoacetica, this enzyme is made up of .alpha. and .beta.
subunits, Moth.sub.--2312 and Moth.sub.--2314, respectively.
However a potential homolog was only found for the .beta.-subunit
and it is not known why C. acetobutylicum would have a functional
.beta.-subunit without an .alpha.-subunit. Interestingly, the
.beta.-subunit homolog in C. difficile is not located near the
.alpha.-subunit, as would be expected. It is possible C. difficile
only needs the one enzyme CD3317 to reduce CO.sub.2 to formate. For
the remaining enzymes in the Eastern branch, good homologs were
found except for one. CAC0291 has poor homology but is a
bifunctional enzyme in C. acetobutylicum, which codes for both the
needed methylenetetrahydrofolate reductase and a homocysteine
S-methyltransferase.
[0054] Unlike the Eastern branch, C. acetobutylicum is missing most
of the enzymes from the Western branch. The only good homologs
which were found in C. acetobutylicum are two carbon monoxide
dehydrogenases. A second potential homolog is a
methyltetrahydrofolate methyltransferase, CAC0578, which has very
poor protein identity with the corresponding enzyme in both M.
thermoacetica and C. difficile. However, CAC0578 is annotated as
being able to catalyze the reaction from methyl-H.sub.4folate to
H.sub.4folate, the reaction that the M. thermoacetica and C.
difficile enzymes carry out. The remaining components of the
Wood-Ljungdahl pathway, the corrinoid iron-sulfate protein (CFeSP)
and the acetyl-CoA synthase, have no homologs in C. acetobutylicum.
It is significant to note that all these homologs identified in C.
acetobutylicum (and all genes belonging in the same operon with
those) are highly expressed throughout the course of the culture of
C. acetobutylicum. These data were obtained from a very detailed
global gene expression study of C. acetobutylicum (54). Thus, we
anticipate that expression of these genes will not be limiting the
ability to institute the W-L pathway in C. acetobutylicum.
[0055] In some embodiments, at least four genes will be cloned into
C. acetobutylicum to generate a complete Wood-Ljungdahl pathway
(Table 1): a formate dehydrogenase (e.g., CD3317, 2.1 kb), the
CFeSP .alpha.-subunit (e.g., CD0726, 1.4 kb), the CFeSP
.beta.-subunit (e.g., CD0725, 0.9 kb), and an acetyl-CoA synthase
(e.g., CD0728, 2.1 kb). In addition, a methyltetrahydrofolate
methyltransferase (e.g., CD0727, 0.8 kb) and the .beta.-subunit of
the formate dehydrogenase (e.g., CD1537, 1.4 kb) may also be
introduced. In some embodiments, these genes are C. difficile
genes, since C. difficile has a complete W-L pathway and is a
closer relative to C. acetobutylicum than M. thermoacetica. In some
embodiments, because of the number and size of the genes, at least
two plasmids are used to express the genes in C. acetobutylicum. In
some such embodiments, in order to facilitate a plurality of
plasmids to exist together in C. acetobutylicum, the different
plasmids comprise different origins of replication. For example, in
some embodiments, one of the expression vectors uses the origin of
replication from the B. subtilis plasmid pIM13, obtained from the
C. acetobutylicum plasmid pIMP1 (55), and the second origin is
derived from the C. butyricum plasmid pCBU2, obtained from the C.
acetobutylicum plasmid pSYL2 (56). In some embodiments, the gene
needed to complete the Eastern branch, CD3317, is placed on one
plasmid under the control of the thiolase (thl) promoter, a strong
clostridial promoter (57-58), while the three genes needed to
complete the Western branch, CD0725, CD0726, and CD0728, are placed
on the second plasmid as an operon under control of the phosphate
butyryltransferase (ptb) promoter, another strong clostridial
promoter (23, 57).
[0056] In some embodiments, engineered strains are grown in serum
bottles using a modified Hungate technique (59) in defined media
(60) under CO.sub.2/H.sub.2. Briefly, small test tubes are filled
with nonsterile, defined media, and gassed using CO.sub.2. After
gassing, a butyl rubber stopper is used to seal the tube and a
crimped metal seal is added. The tube and media are then
autoclaved. Before inoculation, the tubes are filled with H.sub.2
and CO.sub.2 (80:20, v/v) to a final pressure of 0.2 MPa. These
conditions were successfully used for Moorella sp. HUC22-1 (61-62).
Other conditions will be apparent to those of skill in the art and
the invention is not limited in this respect. In some embodiments,
a defined clostridial medium (60) is used with minimal glucose (1
to 80 g/L can be used).
[0057] In some embodiments, CO.sub.2 uptake and utilization in C.
acetobutylicum is monitored during bioprocessing. In some
embodiments, the concentration of CO.sub.2 and H.sub.2 is measured
in the headspace of the anaerobic fermentor throughout the
fermentation via gas chromatography (62), and similar to previously
described methods (63). In some embodiments, the recombinant
strains are able to consume the CO.sub.2 and H.sub.2 while the
wild-type control show minimal to no consumption. A second assay
uses C.sup.13 labeled carbon dioxide, similar to the original Wood
paper investigating the pathway (64). In some embodiments, C.
acetobutylicum is grown on defined media with minimal glucose and
C.sup.13O.sub.2/H.sub.2 pumped into the head space. After the
fermentation, acetate is isolated and run through a mass
spectrometer to determine the relative amount of heavy acetate with
C.sup.13 to non-heavy acetate. In some embodiments, the recombinant
C. acetobutylicum with the W-L pathway is able to produce heavy
acetate, while the control wild-type is not able to produce heavy
acetate.
[0058] In some embodiments, the gene from C. difficile, CD0727, is
added to the Western operon on one of the expression plasmids, to
make an operon of CD0725-CD0726-CD0727-CD0728. In some embodiments,
this expression plasmid is transformed into a C. acetobutylicum
strain harboring only the Eastern expression plasmid to obtain a
strain capable of high rates of CO.sub.2/H.sub.2 consumption. In
some embodiments, the .beta.-subunit of the formate dehydrogenase
from C. difficile will be added to the Eastern expression plasmid
and transformed into a C. acetobutylicum strain harboring the
larger Western expression plasmid. In some embodiments, the genes
related to the Wood-Ljungdahl pathway are cloned from M.
thermoacetica (instead of from C. difficile) and overexpressed in
C. acetobutylicum.
[0059] In some embodiments, to further enhance CO.sub.2 and H.sub.2
utilization by the acetogen, the native CAC genes are overexpressed
in the acetogen (Table 1). In some embodiments, the native
hydrogen-uptake genes (namely: CAC0028--hydA,
CAC0808-0811--hybG-hypE-hypF-hypD, CAC3230--ferredoxin,
CAP0141-0143--mbhS-mbhL-hyaD) are overexpressed in the acetogen
using strong promoters, like the ptb, thl, and the pta
(phosphotransacetylase) promoters.
[0060] In some embodiments, random chemical mutagenesis (65) or
transposon mutagenesis (66-68) is employed to screen for an
acetogenic strain that uses CO.sub.2 and H.sub.2 at high rates. In
some embodiments, the genes that are introduced into the microbe to
generate the Wood-Ljungdahl pathway are integrated into the
microbial chromosome using a markerless technology (69).
Aerobic TAG Production Using Oleaginous Microbes
[0061] In some embodiments, a carbon source, for example, a
carbohydrate source, is converted in an aerobic fermentation
process to a lipid or oil, for example, to a TAG. In some
embodiments, the aerobic fermentation process is carried out by a
microorganism or microbe. In some embodiments, the microorganism is
an oleaginous microorganism, for example, an oleaginous yeast. In
some embodiments, the microorganism is a microorganism described in
U.S. provisional application U.S. Ser. No. 61/309,782, filed Mar.
2, 2010, the entire contents of which are incorporated herein by
reference. In some embodiments, the microorganism is Yarrowia
lipolytica. In some embodiments, the microorganism is a genetically
engineered oleaginous microorganism, for example, a Y. lipolytica
that overexpresses a stearoyl-CoA Desaturase (SCD) gene. In S.
cerevisiae, a stearoyl-CoA desaturase gene was identified as Ole1
in 1990 (Stukey J E, et al., J Biol Chem., 1990, 265(33):20144-9).
The human stearoyl-CoA desaturase gene was partially characterized
in 1994 via isolation of a 0.76 kb partial cDNA from human adipose
tissue (Li et al., Int. J. Cancer, 1994, 57, 50 348-352). The gene
was fully characterized in 1999 and it was found that alternative
usage of polyadenylation sites generates two transcripts of 3.9 and
5.2 kb (Zhang et al., Biochem. J., 1999, 340, 255-264). In S.
cerevisiae, fatty acid monodesaturation is catalyzed by the
endoplasmic reticulum (ER)-resident and essential
.DELTA.9-desaturase, Ole1 (Martin C E, Oh C S, Jiang Y, Regulation
of long chain unsaturated fatty acid synthesis in yeast. Biochim
Biophys Acta. 2007 March; 1771(3):271-85. Epub 2006 Jul. 13. An
exemplary oleaginous yeast overexpressing an SCD gene is depicted
in FIG. 3. Such microbes can utilize various carbon sources for
growth and TAG production, including acetate. FIG. 4 shows that an
exemplary Y. lipolytica overexpressing an SCD gene is able to
thrive at acetate concentrations of 10%, making it an ideal
candidate for the TAG production methods and bioreactors described
herein.
[0062] Y. lipolytica is a non-pathogenic oleaginous yeast that can
use a variety of carbon sources, including organic acids,
hydrocarbons and various fats and oils. The term "oleaginous"
refers to a microbe that can accumulate more than 20% of its dry
cell weight as lipid (see C. Ratledge et al., Microbial routes to
lipids. Biochem Soc Trans. 1989 December; 17(6):1139-41). According
to some aspects of this invention, Y. lipolytica represents a
microbe for biofuel or biofuel precursor production, because Y.
lipolytica is an obligate aerobe with the ability to assimilate
carbohydrates, for example, glucose, or glycerol as a sole carbon
source, and, compared to other yeast strains, Y. lipolytica has a
higher glucose to fatty acid and triacylglycerol (TAG) flux and
higher lipid storage capacity. See, e.g., Beopoulos A, Cescut J,
Haddouche R, Uribelarrea J L, Molina-Jouve C, Nicaud J M, Yarrowia
lipolytica as a model for bio-oil production. Prog Lipid Res. 2009
November; 48(6):375-87. Further, Y. lipolytica is one of the more
intensively studied `non-conventional` yeast species and genome
sequencing, including mitochondrial DNA, of Y. lipolytica was
completed recently. Kerscher S, Durstewitz G, Casaregola S,
Gaillardin C, Brandt U., The complete mitochondrial genome of
yarrowia lipolytica. Comp Funct Genomics. 2001; 2(2):80-90. The
availability of genomic sequence data makes genetic manipulation
more accessible., even though functional annotation of genomic
sequences is not complete. See, e.g., Sokolova L, Wittig I, Barth H
D, Schagger H, Brutschy B, Brandt U., LILBID-mass spectrometry of
protein complexes from blue-native gels, a sensitive top-down
proteomic approach. Proteomics. Published online 2010 Feb. 1, PMID:
20127694.
[0063] Some aspects of this invention relate to an aerobic microbe
engineered and/or optimized for large-scale TAG or TAG precursor
production. In some embodiments, the engineered aerobic microbe
comprises an increased SCD gene product activity. In some
embodiments, the microbe exhibits an increased fatty acid synthesis
rate, an increased TAG storage, and/or an additional required or
desirable trait.
[0064] In some embodiments, the engineered aerobic microbe is an
oleaginous yeast, for example, Y. lipolytica overexpressing an SCD
gene. In some embodiments, the engineered yeast exhibits highly
desirable and unexpected phenotypic characteristics, for example:
increased carbon to oil conversion approaching theoretical values,
robust growth, continuous oil production, remarkable biomass
production, and increased tolerance of the carbon source and
associated substances. In some embodiments, the engineered yeast
provided by aspects of this invention exhibits a carbon to oil
conversion of about 0.025 g/g (g TAG produced/g Glucose consumed),
about 0.5 g/g, about 0.75 g/g, about 0.1 g/g, about 0.15 g/g, about
0.2 g/g, about 0.25 g/g, about 0.29 g/g, or about 0.3 g/g,
approaching theoretical values, continuous oil production. In some
embodiments, the engineered yeast provided by aspects of this
invention exhibits a biomass production that is increased about
2-fold, about 2.5-fold, about 5-fold, about 7.5-fold, about
10-fold, about 15-fold, about 20-fold, about 25-fold, about
30-fold, about 32-fold, about 35-fold, or about 40-fold as compared
to wild type yeast. In some embodiments, the engineered yeast
provided by aspects of this invention exhibits tolerance to the
carbon source and associated substances at concentrations of up to
about 150%, up to about 175%, up to about 200%, up to about 225%,
up to about 250%, up to about 275%, up to about 300%, up to about
325%, up to about 350%, up to about 375%, up to about 400%, or up
to about 500% of that of the highest concentrations tolerated by
wild type yeast.
Bioreactors for TAG Production
[0065] Some embodiments of this invention provide bioreactors for
the production of TAG by biological fermentation. FIGS. 2a and 2b
depict two exemplary bioreactors that include separate aerobic and
anaerobic fermentors for the aerobic conversion of a carbon
substrate to a TAG and the anaerobic fixation of CO.sub.2,
respectively. In some embodiments, an integrated bioreactor system
is provided that comprises an aerobic fermentor for the growth of
an oleaginous microbe and/or TAG production, and an anaerobic
fermentor where fixation of CO.sub.2 and production of a carbon
substrate (e.g., acetate) take place, wherein the CO.sub.2 produced
during growth/TAG production is used for the anaerobic production
of the carbon substrate and/or wherein the carbon substrate (e.g.,
acetate) is used as the carbon source for the aerobic growth/TAG
production (FIG. 2b). In some embodiments, the aerobic fermentation
process uses a carbohydrate feedstock as a carbon source. In other
embodiments, however, no carbohydrate feedstock is used but the
entire bioprocessing is run on CO.sub.2 as the sole carbon source.
In some embodiments, CO.sub.2 is assimilated along with hydrogen or
electrons via a biocathode from an external electric current and
reduced to a reduced carbon substrate by a CO.sub.2 fixing
bacterium, for example, an acetogenic bacterium, that is either
engineered or is natively capable of producing reduced carbon
substrates, e.g. ethanol, acetate or butyrate. In some embodiments,
the carbon substrate is used as a carbon source in the aerobic
fermentor for oil production by an oleaginous microbe. In some
embodiments, the net input is CO.sub.2 and H.sub.2 or electricity,
and the net output is TAG, e.g., oil for biodiesel production. In
some embodiments, part of the CO.sub.2 introduced into the
anaerobic fermentor is recycled from the aerobic TAG-producing
fermentor.
[0066] In some embodiments, the anaerobic fermentor is used to
capture CO.sub.2 produced in an aerobic fermentation process, for
example, in the production of TAG by an oleaginous microbe, and the
captured CO.sub.2 is converted into a biofuel (e.g., ethanol) by
culturing an appropriate Clostridium strain in the presence of the
CO.sub.2 and hydrogen under anaerobic conditions. In some
embodiments, this "CO.sub.2 recycling" yields almost double the
amount of biofuel produced from a certain amount of carbohydrate
feedstock. An exemplary bioreactor according to this concept is
depicted in FIG. 2a.
[0067] The anaerobic fermentors described herein provide anaerobic
conditions, which refers to conditions that are substantially
devoid of oxygen. The aerobic fermentors described herein provide
aerobic conditions, which refers to conditions in which oxygen is
present, abundant, or over-abundant. In some embodiments, the
conditions within the fermentors are monitored prior to and/or
during the bioproces sing carried out to generate TAG as described
herein. For example, in some embodiments, the anaerobic fermentor
comprises a turbidometer to assess the cell density of anaerobic
acetogens; a gas chromatography apparatus to assess CO.sub.2 and
H.sub.2 partial pressures at the input and output of the fermentor;
a mass flow meter to assess the gas flow rate; a pH meter to assess
the acidity of the media; a thermometer to assess the temperature,
and/or an HPLC apparatus to assess the acetate concentration and/or
the concentration of other carbon substrates produced, for example,
of ethanol, butyrate, or organic acids. Similarly, in some
embodiments, the aerobic fermentor comprises a turbidometer to
assess the cell density of the oleaginous microbe, an HPLC
apparatus to assess the concentration of the carbon source (e.g.,
acetate) in medium, or the concentration of other organic compounds
serving as the carbon source, (e.g., citrate, organic acids), a gas
chromatography apparatus to measure oxygen and carbon dioxide
partial pressures at inflow and/or outflow; a mass flow meter to
measure the gas flow rate; a pH meter to measure the acidity of the
media, and/or a thermometer to measure the temperature of the
media. In some embodiments, the fermentors further comprise one or
more controllers that receive input from any combination of the
above listed measuring devices and adjust the respective parameter
to fall within a desired range or to approximate a desired value.
Methods and devices to adjust the parameters indicated above, for
example, heaters and coolers for the regulation of temperature, gas
inflow and outflow valves, etc., are well known to those of skill
in the art, and the invention is not limited in this respect.
[0068] In some embodiments, the bioreactor further comprises a
carbon substrate (e.g., acetate, butyrate, etc.) concentration
device comprising two dialysis units, the first for the extraction
of the carbon substrate from the fermentation medium of the
anaerobic fermentor using an amine such as ALAMINE.RTM. 336 and the
second, containing a caustic solution, for the extraction of the
carbon substrate from the ALAMINE.RTM. 336 solution. This method
can, for example, be used for the concentration of butyrate from
fermentation broths achieving concentrations of butyrate of
approximately 300 g/L.
[0069] In some embodiments, the free cell anaerobic fermentor is
replaced by a packed bed that employs fibers as immobilization
support for the anaerobic acetogens. The purpose of the
introduction of a fiber bed immobilized cell reactor is to increase
the volumetric productivity of acetate production through a
continuous operation without washing out the anaerobic acetogens.
In some embodiments, this approach yields a ten-fold increase in
the volumetric productivity of the acetogen. In some embodiments,
the packed bed fermentor is continuously or semi-continuously
seeded with acetogens from a free-cell culture vessel.
[0070] In some embodiments, the aerobic fermentor of the bioreactor
comprises a microorganism described in U.S. provisional application
U.S. Ser. No. 61/309,782, filed Mar. 2, 2010, the entire contents
of which are incorporated herein by reference. For example, in some
embodiments, the aerobic fermentor comprises a Y. lipolytica that
overexpresses an SCD gene product. In some embodiments, the
anaerobic fermentor of the bioreactor comprises an engineered
CO.sub.2 fixing bacterium, for example, an engineered acetogen,
such as an engineered strain of C. acetobutylicum provided
herein.
[0071] In some embodiments, the aerobic fermentor comprises an rMFC
as described in more detail elsewhere herein to achieve CO.sub.2
fixation using electrons instead of hydrogen.
[0072] In some embodiments, the bioreactor further comprises an
electrolytic cell for electrolytic water splitting for the
production of oxygen and hydrogen as depicted in FIG. 2. In some
embodiments, an electrolytic water-splitting device is used for the
generation of the hydrogen stream of the anaerobic fermentor and
the oxygen super-saturated (e.g., via microbubble formation) stream
of the aerobic fermentor. In some embodiments, the generated
O.sub.2 is directed to the aerobic fermentor and/or the generated
H.sub.2 is directed to the anaerobic fermentor. In some
embodiments, the aerobic fermentation process is carried out in a
liquid in the aerobic fermentor and the O.sub.2 produced by
electrolysis of H.sub.2O is dispersed in micro-bubbles with
increased surface area within the liquid and, hence, much higher
volumetric mass transfer coefficient for oxygen in the aerobic
fermentor than typical spargers can produce. In some embodiments,
this advance contributes to achieving high cell densities (OD 350),
for example, the cell densities that yielded the high oil
concentrations shown in FIG. 3.
[0073] In some embodiments, anaerobic conditions are maintained in
the anaerobic fermentor through purging of oxygen traces using
initially a CO.sub.2 stream and, during operation, a mixture of
CO.sub.2 and hydrogen. In some embodiments, C. acetobutylicum
engineered to exhibit the aerotolerance phenotype by deleting the
peroxide repressor (PerR)-homologous protein are employed as the
CO.sub.2 fixing microbes of the anaerobic fermentor.
Biocathodes and Reverse Microbial Fuel Cells (rMFC)
[0074] Some aspects of this invention are based on the recognition
that recent developments in Microbial Fuel Cells suggest that
extracellular electron transfer (EET) can occur from a biocathode
to a microbial culture and provide the reducing equivalents needed
for CO.sub.2 reduction in a reverse MFC configuration. Various
strains of Clostridia have been found capable of catalyzing the
oxidation of reduced organic compounds with the concomitant
generation of electrons, transferred via an anode to an external
circuit of a traditional microbial fuel cell (MFC). Most MFC
research has focused on microbes capable of donating electrons to
the anode from degradation of organic matter. Some embodiments of
this invention provide a reverse operation at the anode, where
electrons from an external source (e.g. a battery) act as reducing
agents of an oxidized compound, such as CO.sub.2, through the
action of microorganisms harboring the relevant (W-L) pathway, and
thus capable of carrying out the reduction reactions. Such
embodiments are supported by recent studies where abiotic cathodes
were replaced with bio-cathodes, in which enzymes enhance reduction
catalysis (24).
[0075] The efficacy of a biocathode in any bioelectrochemical
system (BES) is typically directly related to the current density
(amps per unit area) and coulombic efficiency (% of electrons
recovered by the target product). Notably, as microorganisms in a
biocathode evolve strategies that lower the cathode overpotentials,
higher current densities can be obtained. Typically, autotrophic
microorganisms are believed to be the key players on biocathodes
since the cathode acts as the electron donor for metabolism. As a
consequence, CO.sub.2 (or bicarbonate) can be reduced (or fixed),
though to date there are few studies that directly examine
biocathode carbon reduction as described elsewhere herein.
[0076] Several studies have examined microbial diversity on MFC
cathodes. In one study (25) it was observed that Pseudomonas
flourescens constituted nearly 50% of the microbial load on a
microbial fuel cell deployed in the ocean. In another study,
.alpha.- and .gamma.-Proteobacteria were identified as the dominant
"phylotypes" enriched on the biocathodes under nitrogen limited
conditions (26). Rabaey and co-workers also found enrichment of
Bacteroidetes, .beta.- and .gamma.-Proteobacteria from mixed
sediment-sludge inoculums (27).
[0077] Few studies to date have directly examined the linkage
between cathode electron donation and microbial metabolic
processes. Electron donation from electrodes has been shown to
support and stimulate denitrification, methanogenesis, perchlorate
reduction, and other reductive processes. Denitrification is a
microbially mediated process in which microorganisms respire an
inorganic electron donor while using nitrate, nitrite, nitric oxide
or nitrous oxide as electron acceptor (28). In 1966, the use of a
nitrate reducing biocathode was proposed (29). However, it was not
until 2004 that Gregory and co-workers demonstrated for the first
time the ability of microbial isolates, enriched from a sediment,
to retrieve electrons directly from a cathode (poised at
approximately -0.300 V vs. a standard hydrogen electrode) for
nitrate to nitrite reduction (30). This was an enrichment of
.gamma.-proteobacteria, Gram-positive bacteria and Geobacter-like
phylotypes (.delta.-proteobacteria). Indeed, a pure culture of
Geobacter metallireducens was shown to reduce nitrate to nitrite by
a cathode poised at -0.300 V vs. SHE. When different sludge and
sediment mixtures were used as inocula for a denitrifying
biocathode, the complete denitrification from nitrate to N.sub.2
was observed (31). Notably, for denitrifying biocathodes, the
equilibrium cathode potential is typically around 0V vs. SHE.
[0078] Perchlorate reduction with the help of an anoxic biocathode
was first demonstrated in the presence of 2,6-anthraquinone
disulfonate (AQDS), an extra cellular electron mediator (32).
Perchlorate is widely used as a propellant in the aerospace and
defense industries, and is of environmental concern due to its high
mobility and inhibiting effect on thyroid function (33). Recently,
a novel strain was isolated with the ability to reduce perchlorate
at -0.300 V vs. SHE in the absence of AQDS (33). Moreover, Rozendal
and co-workers suggested the possibility of methanogenesis by
methanogens in a biocathode as well as hydrogen production (34).
Recently, a biocathode biofilm dominated by Methanobacterium
palustre was shown to produce methane in a direct way without
hydrogen as an intermediate (35). Since biocathodes appear to be
able to produce hydrogen and methane in a direct way, biocathodical
acetogenesis is also very likely (36). The reduction of fumarate to
succinate has also been observed by Geobacter sulfurreducens (30)
and Geobacter lovleyi (37) on graphite cathodes, in the absence of
hydrogen or externally added electron mediators. Finally, Geobacter
sulfurreducens was also shown to reduce uranium (VI) to relatively
insoluble uranium (IV) in an anaerobic biocathode poised at
approximately -0.300 V vs. SHE (38). In all cases the means by
which these microbes harvest electrons from the cathodes remains
unknown.
[0079] In some embodiments of this invention, metabolic reduction
of CO.sub.2 is achieved on a biocathode of a reverse microbial fuel
cell (rMFC). In some embodiments, the rMFC provides electrons to a
CO.sub.2 fixing bacterium, e.g., an acetogen, for the reduction of
CO.sub.2 to a carbon substrate. In some embodiments, the rMFC
comprises an amperemeter to measure the current supplied, a
turbidometer to measure the density of the cells in the rMFC,
and/or a gas chromatography apparatus to measure the partial
pressure of CO.sub.2 at the inflow and/or the outflow.
Bioelectrochemical System Architecture
[0080] MFCs are a type of bio-electrochemical reactors that allow
one to harness energy (as electrical current) from microbial
metabolism. MFCs typically consist of an anaerobic chamber that
houses the anode, a fuel (e.g. compost or wastewater) and
associated microbes. The MFC's aerobic chamber houses the cathode
and, in some cases, associated microbes. At the anode, microbes
oxidize organic matter using the electrode as the oxidant, and
hence transfer electrons to the anode. At the cathode, reactions
such as the formation of water from protons and oxygen balance the
oxidation reactions at the anode. Most studies to date have focused
on harnessing electricity from microbial degradation of organic
matter in the anode chamber, with the intention of using MFCs for
alternative energy generation. However, many studies have shown
that the coulombic efficiency (the percentage of electrons
available in the fuel that are captured by the anode) of anodic
power generation is low when complex fuels are used. Given the
limitations of MFC power production, a number of researchers
believe that the greatest potential of such bioelectrochemical
systems (or BESs) is for the generation of fuels and other
commodities (39). For example, investigators have recently
developed a BES that generates hydrogen gas at the cathode (40-41).
In this reactor, bacteria at the anode oxidize carbon substrates,
generating protons and electrons as in a MFC. However, in lieu of
providing oxygen to the cathode, current is supplied from an
external power source, stimulating the production of hydrogen.
Since the protons and electrons are being derived from organic
matter through biocatalysis, the voltage needed to generate H.sub.2
is an order of magnitude lower than that needed for electrolysis of
water. For example, this system uses the equivalent of 0.2 mol
hydrogen energy per mole of hydrogen produced, compared to the 1.7
mol loss typical of electrolysis (41). A variety of other products
have already been generated using BESs including glutamic acid
(42), propionic acid (43), succinate (44), sulfur (45), methane
(46), formate (47), and ethanol (48).
[0081] Electricity for embodiments using a biocathode or rMFC can
be supplied by various means, including combustion of biomass or
municipal waste that can also generate the CO.sub.2 used in the
process.
[0082] The functions and advantages of these and other embodiments
of the present invention will be more fully understood from the
example section below. The following examples section is intended
to illustrate the benefits of the present invention and to describe
particular embodiments, but does not exemplify the full scope of
the invention. Accordingly, it will be understood that the examples
section is not meant to limit the scope of the invention.
EXAMPLES
Example 1
Aerobic TAG Production Using Engineered Microbes
[0083] In some embodiments, the engineered microbe used for aerobic
carbon source to TAG fermentation is capable of converting, at
almost maximum theoretical yields, carbohydrates to oils and fats
for biodiesel production (see FIG. 3). In some embodiments, the
oleaginous microbe is a Y. lipolytica overexpressing a stearoyl-CoA
desaturase (SCD) gene, which has been identified as a key regulator
of carbohydrate to lipid conversion. In some embodiments, the
oleaginous microbe comprises an increased activity of an SCD gene
product. In some embodiments, the oleaginous microbe further
comprises a genetic modification that increases expression of one
or more genes chosen from the group of Hemoglobin, Cytochrome,
GLUT, Malic Enzyme, ACC, SCD, FAA1, ACS, ACS2, FAT1, FAT2, PCS60,
ACLY, FAS, Acyl-CoA synthetase, Pyruvate carboxylase, and AMPK
genes, and/or a genetic modification that reduces expression of a
JNK2 gene. In some embodiments, the oleaginous microbe is
engineered to expresses a native gene as described above under the
control of a strong, heterologous promoter. In some embodiments,
the oleaginous microbe expresses a heterologous gene as described
above, for example, a mammlian (e.g., a murine or human) SCD,
hemoglobin, cytochrome, GLUT, ME, etc., gene under the control of a
constitutive promoter. For example, in some embodiments, a Y.
lipolytica expressing a murine or human SCD gene under the control
of a constitutive promoter is employed.
[0084] In some embodiments, concentrations between 80-100 g/L of
biodiesel are achieved within 3 days in aerobic fermentors at
conversion yields of .about.0.26-0.29 grams of biodiesel per gram
of glucose consumed. The stoichiometry of the oil synthesis pathway
of the aerobic fermentor (assuming tripalmitin to be representative
of the oil composition) is:
14C.sub.6H.sub.12O.sub.6+11.5O.sub.2.fwdarw.1C.sub.51H.sub.98O.sub.6+33C-
O.sub.2+15.5ATP (Eq. 2)
[0085] If the ATP produced in the above overall reaction were to be
used for the fixation of CO.sub.2 this would reduce the glucose
requirement by only 0.25 moles, indicating that oil (tripalmitin)
synthesis is already energetically optimized. This performance
suggests that biodiesel can be produced at a cost ranging from
$1.50-2.50/gallon depending on the feedstock used. Current research
aims at optimizing the use of different feedstocks as substrates
for the oleaginous microbe along with the corresponding downstream
operations associated with each feedstock. However, the capability
of the engineered oleaginous microbe to utilize glucose (in pure
form or mixtures from cellulosic/hemicellulosic biomass
hydrolysates), crude glycerol, ethanol, acetate, and butyrate as a
carbon source has been established.
[0086] In the processing scheme depicted in FIG. 2b the "product of
CO.sub.2 fixation" is used for the growth of the oleaginous microbe
and oil production. In some embodiments, this product is acetate.
The reason to use acetate is two-fold: First, acetate is the
product of most acetogens fixing anaerobically CO.sub.2, hence
there is significant prior knowledge on this topic, along with the
genes and molecular constructs that are required to further
modulate the acetogenic W-L pathway (FIG. 1). The second reason is
that the oleaginous organism can readily metabolize acetate and
produce TAG. FIG. 4 shows a time course for the growth of the
oleaginous Y. lipolytica overexpressing SCD on 5% and 10% acetate.
It can be seen that growth was uninhibited even at acetate levels
of greater than 10%. The growth of the mutant was observed to be at
least 3-fold greater than the parental strain. Oil production by
the mutant was similarly 3-fold greater than that of the parental
strain.
Example 2
Microbial Conversion of H.sub.2/CO.sub.2 to Acetate
[0087] Production of acetate from H.sub.2/CO.sub.2 proceeds through
the Wood-Ljungdahl pathway. This metabolic pathway has been
well-studied (71). Numerous groups have isolated various acetogens
which can produce acetate (72-74). These acetogens produce acetate
according to the following equation:
4H.sub.2+2CO.sub.2.fwdarw.CH.sub.3COOH+2H.sub.2O (Eq. 3)
[0088] Most acetogens produce acetate at almost theoretical yields.
While there has been much research in understanding the mechanisms
behind this pathway, there has been very little research since the
1980's on improving the rate of acetate production by these
organisms. As an example, FIG. 6 shows growth and acetate
production characteristics of an acetogenic strain (Acetobacterium
woodii) researched in the 1970's (72).
[0089] The mass yield of this stoichiometry is calculated to be
7.375 g acetate/g H.sub.2. From the graph, an acetate productivity
of approximately 2.5 g/L/day can be estimated. At this productivity
and titer, it is estimated that 213 M gallons of liquid culture
would be necessary to produce enough acetate to be processed into
50 million gallons of biodiesel per year (see Table 2). In a
typical production plant, this would require 213, 1M-gallon
fermentors, a 25-30-fold increase relatively to ethanol
fermentation processes with comparable annual output. At this
productivity, therefore, the process would be economically
infeasible. However, one should note the exceptionally low cell
densities of these fermentations, approximately 25-30 fold lower
than the OD of typical ethanol fermentations routinely run with the
acetogens described herein. This indicates that the specific rate
of CO.sub.2 fixation and acetate production by the anaerobic
bacteria of FIG. 6 are comparable to those of another anaerobic
process, ethanol fermentation. Therefore, it is very likely that
the total volumetric productivity of acetate production by
anaerobic acetogens can be significantly increased by following two
strategies: (1) increasing the cell density of the anaerobic
fermentor while maintaining the same specific productivity, and,
(2) by increasing the specific CO.sub.2 fixation rate and rate of
acetogenesis through metabolic engineering aiming at increasing the
W-L pathway flux.
TABLE-US-00002 TABLE 2 Scale and Efficiency Calculations Basis
Running Days 300 days/year Production of biodiesel 50,000,000
gallons/year 189,250,000 Liters/year 166,540,000 kg biodiesel/year
555,133 kg biodiesel/day Calculations Amount of acetate
required.sup.a 2,022,419 kg acetate/day Acetogen culture volume
required 1,011,209,418 L 267,162,330 gallons Number of 1M
Fermentors needed 267 Fermentors Productivities Yields.sup.c
Conversions Acetogenesis (Acetate) 0.28 g oil/g glucose 3.785
Liters/gallon 2.0 g/L/day.sup.b 0.274 g oil/g acetate 0.88 kg
biodiesel/Liter 143 MJ/kg H.sub.2 42.2 MJ/kg biodiesel
Hydrogen-to-Oil Energy Efficiency H2 to Acetate Conversion 7.375 g
acetate/g H.sub.2.sup.d Acetate to Oil Conversion 0.274 g oil/g
acetate.sup.c 2.024 g oil/g H.sub.2 Energy Efficiency.sup.e 59.74%
Total Overall Photon-to-Oil Efficiency Reference PEC Efficiency
(Light to H2) 12.0% (76) Total Overall Photon Efficiency.sup.f
7.17% PV Efficiency (Light to Electricity) 25.0% (77) Electrolyzer
Efficiency (Electricity to H2) 67.0% (78) Total Overall Photon
Efficiency.sup.f 10.01% .sup.aAssuming complete transesterification
of oil to biodiesel, and no losses in acetate separation
.sup.bReport of 10 g/L in 5 days (71) .sup.cTheoretical yields
based on stoichiometric calculations .sup.d4 H.sub.2 + 2 CO.sub.2
.fwdarw. CH.sub.3COOH + 2 H.sub.2O (13) .sup.eDefined as (liquid
fuel energy out)/(hydrogen energy in) .sup.fDefined as (liquid fuel
energy out)/(photon energy in)
[0090] For increasing the functional cell mass, since growing
biomass from hydrogen is not the primary purpose of this process,
cells can be grown to a high density using some easily
metabolizable growth substrate (e.g., glucose), and then the cells
can be used in stationary phase to continuously convert CO.sub.2
and H.sub.2 into acetate, thus addressing the problem of low titers
dictating excessive culture volumes for adequate production.
Methods for improving acetogenic CO.sub.2 fixation by implementing
or increasing flux through the W-L pathway in acetogens are
described elsewhere herein. Both approaches result in increased
acetate titers.
Example 3
Acetate to Oil Stoichiometric Calculations
[0091] The theoretical yield of de novo synthesis of
triacylglyceride from acetate was estimated using a carbon chain
pivot method (75). Intermediate metabolites (e.g. glucose,
pyruvate, acetyl-CoA) are used as central carbon pivots in balance
equations involving segments of metabolic pathways. In order to
obtain the stoichiometry for an entire metabolic pathway, balance
equations are combined such that the intermediate carbon pivots sum
to zero. After summation, the remaining non-pivot metabolites are
either inputs (negative) or outputs (positive) of the pathway. This
method can account for energetics as well by including co-factors
such as ATP, NADH, NADPH in the pivot table and constraining these
to positive values. The calculation of the stoichiometry for the
conversion of glucose into tripalmitin (representative lipid) uses
seven balance equations, which eliminates pyruvate, acetyl-CoA,
NADH and NADPH from the total balance, to produce the final
stoichiometry (shown in Example 4). Acetate is converted into
acetyl-CoA, which is the precursor for both fatty acid elongation
and cellular respiration. NADPH is generated in the
Transhydrogenase Cycle (76).
49CH.sub.3COOH+13.5O.sub.2.fwdarw.1C.sub.51H.sub.98O.sub.6+47CO.sub.2+15-
ATP (Eq. 4)
[0092] It is observed that carbon flux is roughly split between
anabolic and catabolic pathways, highlighting the energy intensive
nature of lipid synthesis. On a mass basis, for 1 kg of tripalmitin
produced, 3.64 kg of acetate is consumed and 2.5 kg of carbon
dioxide is respired. This represents a theoretical mass yield of
0.274 g oil/g acetate. This is comparable to a calculated
theoretical yield on glucose at 0.32 g oil/g glucose. The remaining
hydrogen and oxygen (not shown in the equation) is in the form of
water and reducing equivalents.
Example 4
Calculation of Overall Energy Efficiency
[0093] If it is assumed that hydrogen is the limiting factor, the
yields of both bioprocesses (aerobic and anaerobic) can be combined
and an overall yield for the process of producing oil from hydrogen
can be obtained. On a mass basis the theoretical yield is 2.02 g
oil/g H.sub.2. This can be converted to an energy basis, according
to the energy densities of biodiesel and hydrogen. The overall
energy efficiency for our process, defined as energy content of
fuel produced divided by energy content of hydrogen consumed, is
59.74% (see Table 2).
[0094] To facilitate comparison between other technologies, the
biodiesel production scheme can be expanded to encompass processes
for the production of hydrogen from sunlight. Two possible
processes appear amenable: (1) direct hydrogen generation from
sunlight via photo-electrochemical cells (PEC), (2) photovoltaic
(PV) conversion of sunlight to electricity followed by the
electrolysis of water to produce oxygen and hydrogen. PEC units can
reach 12% efficiency under certain configurations (77). For PV, 40%
efficiency is achievable, although 25% is much more common (78).
Hydrolysis of water using an electrolyzer is estimated to have an
energy efficiency of 67% (79). If we combine these efficiencies
with the biodiesel production scheme, we obtain theoretical
sunlight-to-biodiesel efficiencies of 7.17% for PEC and 10.01% for
PV-hydrolysis (assuming 25% PV efficiency). PV-hydrolysis is the
more efficient process. These theoretical efficiencies compare
favorably to schemes dependent on photosynthesis that have
efficiencies in the ranges of 0.1%-2% for plants and 2-6% for
microalgae (80).
Example 5
Exemplary Process Flow Diagram
[0095] Possible flow rates can be estimated for implementation by
incorporating the theoretical stochiometries into a process scheme,
and performing a mass balance over the process (FIG. 7). A basis of
500 kg/hr oil production was used, which roughly translates into a
small 1M gallon/yr oil production plant (note that water streams
were omitted from the process diagram, and the stoichiometric
equation for oil production from acetate also omits the production
of water and reducing equivalents). The carbon dioxide recycled
from the aerobic reactor contributes to half the total carbon
dioxide demand in the anaerobic reactor. For the hydrolysis of
water to form hydrogen and oxygen, an electric-to-hydrogen energy
conversion efficiency of 67% (79) was used. Since there are only a
few inputs to the process, the electricity consumed for
electrolysis can give perspective to the level of power demand
required for the process. 14.4 MW can reasonably be supplied by 3-4
wind turbines; a single wind turbine supplying about 3-5 MW
each.
Example 6
Development of the Biocathode Screening Protocol and Construction
of rMFC
[0096] Recent studies (70) have shown that electrode potential
significantly influences microbial community population dynamics,
rates of EET and coulombic efficiency. Moreover, it was shown that
active potentiostatic control of the electrodes can be used to
select for (or possibly evolve) microbes with higher rates of EET
and higher coulombic efficiencies (unpublished data), so active
potentiostatic control is necessary to conduct robust, repeatable
assessments of microbial bioelectrochemical activity.
[0097] In some embodiments, coupling of active potentiostatic
measurement and control with a multi-well MFC (96 electrodes) is
implemented to: a) screen for bioelectrical activity among strains,
b) determine the electrode properties (e.g. potential, duty
cycling) that are optimal for current production, and, c) identify
strains with optimized current generation and coulombic efficiency.
The 96-well MFC allows to ally substrate utilization with electron
acceptance, end product production and microbial population growth.
In concert with the electrochemical measurements, this approach
provides the most comprehensive view of microbial metabolism (EE T,
metabolic rates) in relation to bioelectrochemical attributes.
[0098] In some embodiments, the fermentors consist of three primary
components, a top, a mid, and bottom plate (FIG. 5). In some
embodiments, the top plate is fabricated of PEEK (chemically and
biologically non-reactive) and milled to allow a "1/4-28 thread"
gastight HPLC fittings to be secured above each well of the glass
plate. The fittings allow to sample fluids for microbial
characterization, fluid characterization and dissolved gas
analyses. In some embodiments, the top plate comprises a 1.5 mm
hole to accommodate an insulated titanium wire. This wire can be
potted in place and a small graphite electrode can be affixed to
the wire using silver epoxy. This can serve as the cathode. In some
embodiments, the mid-section of the bioreactor consists of a
commercially available high strength glass 96-well microtitre plate
(Rapp Polymer GmbH, Germany). These plates are available in several
volumes (with different heights), from 1.5 .mu.L to 1.5 mL. The
appropriate size can be selected as needed. High-strength glass is
ideal as it is cost-effective and electrically insulated. In some
embodiments, the bottom plate of the bioreactor is fabricated of
non-conductive machinable ceramic, and engineered to include a low
gas permeability proton exchange membrane that physically separates
each well (Nafion-PTFE 30%). The bottom plate can also include a
flow-through channel that houses another larger electrode (about
the length and width of the assembly). This electrode can be used
for hydrolysis, and the subsequent hydrogen ions can diffuse across
the membrane to support biosynthesis. The flow through channel can
be flushed at a rate sufficient to insure steady state. Based on
calculations, this bioreactor can be gastight and capable of
withstanding 500 PSI (ca. 34 atm) up to 110.degree. C. This modular
system represents the first bioreactor design that can be used to
study aerobic or anaerobic microbial strains capable of EE T, while
keeping substrates and volatiles contained within the well, in a
high-throughput format. In sum, this configuration will allow to a)
interrogate up to 96 strains at a time, b) maintain each strain in
well-defined conditions, c) poise each cathode independently by
using a single-channel potentiostat and 12-channel multiplexer
(Gamry Inc.), and d) ally microbial growth to electron acceptance
and end product production.
[0099] In some embodiments, to screen for bioelectrical activity,
select strains and mutants are placed into each well, in
appropriate media and substrates. In some embodiments, strains will
be run in quadruplicate for statistical robustness. In some
embodiments, the gas headspace of each well is flushed with a
CO.sub.2:N.sub.2 mix to achieve appropriate dissolved inorganic
carbon concentrations and pH. When ready, the potentiostat is be
configured to poise all 96 wells at a pre-determined potential. In
some embodiments, experiments are run for up to 84 hours. This
supports microbial growth within the wells and allows the
establishment of active biofilms on the electrode. In some
embodiments, upon completion of the cycle, a fluid and gas
headspace sample is manually collected from each well and analyzed
for changes in total inorganic carbon via gas chromatography and
for end product production using, for example, a gas chromatograph
outfitted to extract gasses from aqueous phases, and capable of
quantifying carbon dioxide, oxygen, nitrogen, sulfide, methane, and
carbon monoxide.
[0100] To be considered for further characterization, an isolate
must: a) demonstrate bioelectrical activity; b) measurably reduce
the inorganic carbon concentration in the well, c) produce a
measurable quantity of the desired end product (acetate). In some
embodiments, a strain satisfying these criteria is subjected to
another round of testing. In some embodiments, each such strain is
loaded into a total of 24 wells prepared as described above. To
determine optimal potential for electron acceptance, potentiometry
is used to identify the optimal potential for microbial electron
acceptance for each strain. In some embodiments, to enable robust
quantification of coulombic efficiency and to constrain reaction
kinetics, each select strain is again be grown in 24 wells. Four
wells are subject to the pre-determined potential for one hour. The
multiplexer then subjects the next four wells to a different
potential for two hours and so on, up to six hours at potential
(this can be done sequentially, so that the total incubation time
is 21 hours). These measurements (chrono-amperometry) provide a
time-course of carbon reduction and biofuel production, enabling
robust quantification of coulombic efficiency, as well as reaction
kinetics. In some embodiments, media from each strain are also
subjected to cyclic voltammetry to potentially identify any
redox-active electron shuttles.
[0101] In some embodiments, the identified strains are further
tested using a medium-scale reverse MFC (rMFC). For example, this
reactor can be approximately two liters in volume, consisting of
two glass reactors separated by a proton exchange membrane (White
et al 2009; Reimers et al 2007). Briefly, the reactor embodies the
same principles as above but also includes a distinct chamber for
anode and cathode. The exemplary rMFC uses 24.times.1 cm diameter
rods to form an electrode array. The rMFC can be inoculated with a
target strain, and can be subjected to the same tests described in
the higher resolution screening above. In addition, stable
isotopically-labeled precursors (e.g., .sup.13C bicarbonate) can be
used during the course of the incubations to trace the fate of
inorganic carbon.
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[0182] All publications, patents and sequence database entries
mentioned herein, including those items listed below, are hereby
incorporated by reference for the teachings referenced herein as if
each individual publication or patent was specifically and
individually indicated to be incorporated by reference. In case of
conflict, the present application, including any definitions
herein, will control.
EQUIVALENTS AND SCOPE
[0183] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. The scope of the present invention is not intended to be
limited to the above description, but rather is as set forth in the
appended claims.
[0184] Articles such as "a," "an," and "the," as used herein, may
mean one or more than one unless indicated to the contrary or
otherwise evident from the context. Claims or descriptions that
include "or" between one or more members of a group are considered
satisfied if one, more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process unless indicated to the contrary or otherwise evident
from the context. The invention includes embodiments in which
exactly one member of the group is present in, employed in, or
otherwise relevant to a given product or process. The invention
also includes embodiments in which more than one, or all of the
group members are present in, employed in, or otherwise relevant to
a given product or process.
[0185] Furthermore, it is to be understood that the invention
encompasses all variations, combinations, and permutations in which
one or more limitations, elements, clauses, descriptive terms,
etc., from one or more of the claims or from relevant portions of
the description is introduced into another claim or another portion
of the description. For example, any claim that is dependent on
another claim can be modified to include one or more limitations
found in any other claim that is dependent on the same base claim.
Furthermore, where the claims recite a composition, it is to be
understood that methods of using the composition for any of the
purposes disclosed herein are included, and methods of making the
composition according to any of the methods of making disclosed
herein or other methods known in the art are included, unless
otherwise indicated or unless it would be evident to one of
ordinary skill in the art that a contradiction or inconsistency
would arise.
[0186] Where elements are presented as lists, it is to be
understood that each subgroup of the elements is also disclosed,
and any element(s) can be removed from the group. It is also noted
that the term "comprising" is intended to be open and permits the
inclusion of additional elements or steps. It should be understood
that, in general, where the invention, or aspects of the invention,
is/are referred to as comprising particular elements, features,
steps, etc., certain embodiments of the invention or aspects of the
invention consist, or consist essentially of, such elements,
features, steps, etc. For purposes of simplicity those embodiments
have not been specifically set forth in haec verba herein. Thus for
each embodiment of the invention that comprises one or more
elements, features, steps, etc., the invention also provides
embodiments that consist or consist essentially of those elements,
features, steps, etc.
[0187] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and/or the understanding of one of
ordinary skill in the art, values that are expressed as ranges can
assume any specific value within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates otherwise.
It is also to be understood that unless otherwise indicated or
otherwise evident from the context and/or the understanding of one
of ordinary skill in the art, values expressed as ranges can assume
any subrange within the given range, wherein the endpoints of the
subrange are expressed to the same degree of accuracy as the tenth
of the unit of the lower limit of the range.
[0188] In addition, it is to be understood that any particular
embodiment of the present invention may be explicitly excluded from
any one or more of the claims. Where ranges are given, any value
within the range may explicitly be excluded from any one or more of
the claims. Any embodiment, element, feature, application, or
aspect of the compositions and/or methods of the invention, can be
excluded from any one or more claims. For purposes of brevity, all
of the embodiments in which one or more elements, features,
purposes, or aspects is excluded are not set forth explicitly
herein.
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