U.S. patent application number 12/877785 was filed with the patent office on 2011-01-06 for production and secretion of glucose in photosynthetic prokaryotes (cyanobacteria).
This patent application is currently assigned to Board of Regents, The University of Texas System. Invention is credited to R. Malcolm Brown, JR., David R. Nobles, JR..
Application Number | 20110003345 12/877785 |
Document ID | / |
Family ID | 39275233 |
Filed Date | 2011-01-06 |
United States Patent
Application |
20110003345 |
Kind Code |
A1 |
Nobles, JR.; David R. ; et
al. |
January 6, 2011 |
Production and Secretion of Glucose in Photosynthetic Prokaryotes
(Cyanobacteria)
Abstract
The present invention includes compositions and methods for
making and using an isolated cyanobacterium that includes a portion
of an exogenous bacterial cellulose operon sufficient to express
bacterial cellulose, whereby the cyanobacterium produces
extracellular glucose. The compositions and methods of the present
invention may be used as a new global crop for the manufacture of
cellulose, CO.sub.2 fixation, for the production of alternative
sources of conventional cellulose as well as a biofuel and
precursors thereof.
Inventors: |
Nobles, JR.; David R.;
(Austin, TX) ; Brown, JR.; R. Malcolm; (Austin,
TX) |
Correspondence
Address: |
CHALKER FLORES, LLP
2711 LBJ FRWY, Suite 1036
DALLAS
TX
75234
US
|
Assignee: |
Board of Regents, The University of
Texas System
Austin
TX
|
Family ID: |
39275233 |
Appl. No.: |
12/877785 |
Filed: |
September 8, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11866872 |
Oct 3, 2007 |
7803601 |
|
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12877785 |
|
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60849363 |
Oct 4, 2006 |
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Current U.S.
Class: |
435/100 ;
435/101; 435/161; 435/257.2; 435/72 |
Current CPC
Class: |
C12P 7/10 20130101; Y02E
50/17 20130101; C12P 19/02 20130101; C12P 19/04 20130101; Y02E
50/16 20130101; Y02E 50/10 20130101 |
Class at
Publication: |
435/100 ;
435/257.2; 435/72; 435/101; 435/161 |
International
Class: |
C12P 19/12 20060101
C12P019/12; C12N 1/13 20060101 C12N001/13; C12P 19/00 20060101
C12P019/00; C12P 19/04 20060101 C12P019/04; C12P 7/06 20060101
C12P007/06 |
Claims
1. An isolated cyanobacterium comprising a portion of an exogenous
bacterial cellulose operon sufficient to express bacterial
cellulose, whereby the cyanobacterium produces extracellular
glucose.
2. The cyanobacterium of claim 1, wherein the cyanobacteria is
further defined as producing extracellular glucose and cellulose in
the form of monosaccharides, disaccharides, oligosaccharides or
polysaccharides from photosynthesis.
3. The cyanobacterium of claim 1, wherein the cyanobacteria is
further defined as making monosaccharides, disaccharides,
oligosaccharides or polysaccharides that comprise glucose and
cellulose.
4. The cyanobacterium of claim 1, wherein the cyanobacterium
comprises Synechococcus sp. PCC 7002, Synechococcus leopoliensis
strain UTCC 100, Agmenellum quadruplicatum UTEX B2268, Nostoc spp.,
Anabaena spp., Cyanothece spp., Trichodesmium spp. and
Synechococcus sp. ATCC 27264.
5. The cyanobacterium of claim 1, wherein the glucose, the
cyanobacterial extracellular polysaccharides or both are further
processed as a renewable feedstock for biofuel production.
6. The cyanobacterium of claim 1, wherein the cyanobacterium can
fix CO.sub.2 while producing cellulose and reducing atmospheric
CO.sub.2.
7. The cyanobacterium of claim 1, wherein the cyanobacterium
increases the extracellular production of monosaccharides,
disaccharides, oligosaccharides or polysaccharides upon exposure to
acidic conditions.
8. The cyanobacterium of claim 1, wherein extracellular glucose is
exuded from cells or released from extracellular polysaccharides by
the actions of one or more endogenous secreted glycosyl
hydrolases.
9. An isolated cyanobacterium, comprising: a Synechococcus sp.
comprising a portion of an exogenous bacterial cellulose operon
sufficient to express bacterial cellulose, whereby the
cyanobacterium is capable of producing extracellular
monosaccharides, disaccharides, oligosaccharides or polysaccharides
comprising glucose.
10. The cyanobacterium of claim 9, wherein the cyanobacteria is
further defined as producing extracellular glucose in the form of
monosaccharides, disaccharides, oligosaccharides or polysaccharides
from photosynthesis.
11. The cyanobacterium of claim 9, wherein the cyanobacterium is
further defined as making monosaccharides, disaccharides,
oligosaccharides or polysaccharides that comprise glucose and
cellulose.
12. The cyanobacterium of claim 9, wherein the cyanobacterium
comprises Synechococcus sp. PCC 7002, Synechococcus leopoliensis
strain UTCC 100, Agmenellum quadruplicatum UTEX B2268, Nostoc spp.,
Anabaena spp., Cyanothece spp., Trichodesmium spp., and
Synechococcus sp. ATCC 27264.
13. The cyanobacterium of claim 9, wherein the cellulose, the
cyanobacterial extracellular polysaccharides or both are further
processed as a renewable feedstock for biofuel production.
14. The cyanobacterium of claim 9, wherein extracellular glucose is
exuded from cells or released from extracellular polysaccharides by
the actions of one or more endogenous secreted glycosyl
hydrolases.
15. A method of producing monosaccharides, disaccharides,
oligosaccharides or polysaccharides comprising glucose, comprising:
modifying a cyanobacterium with a portion of an exogenous bacterial
cellulose operon sufficient to express and produce extracellular
glucose; growing the cyanobacteria under conditions that promote
extracellular glucose production; and exposing the cyanobacteria to
an acidic condition, wherein the acid increases extracellular
glucose production and optionally that the viable cells are
returned to the growth medium after glucose harvest for continued
production of cells, biomass, cellulose or glucose.
16. The method of claim 15, further comprising the step of
processing the glucose into ethanol.
17. The method of claim 15, wherein the glucose is used as a
renewable feedstock for biofuel production.
18. The method of claim 15, wherein the cyanobacterium fixes
CO.sub.2 and thus atmospheric CO.sub.2.
19. The method of claim 15, wherein the glucose is used as a
renewable feedstock for animals.
20. A method of fixing carbon into a photobiomass comprising:
growing a cyanobacterium comprising a portion of an exogenous
bacterial cellulose operon sufficient to make cellulose and to
produce extracellular glucose in a CO.sub.2-containing growth
medium; generating glucose with said cyanobacterium, wherein
CO.sub.2 is fixed into glucose at a level higher than an unmodified
cyanobacterium; and calculating the amount of CO.sub.2 fixed into
the glucose to equate to one or more carbon credit units.
21. The method of claim 20, wherein at least one other carbon is
fixed into glucose and at least one other carbon's is equated to
carbon credit units that is included in the calculation.
22. An isolated cyanobacterium comprising a portion of an exogenous
bacterial cellulose operon sufficient to express bacterial
cellulose, whereby the cyanobacterium is capable of producing
extracellular monosaccharides, disaccharides, oligosaccharides or
polysaccharides.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates in general to the field of
exogenous gene expression, and more particularly, to the expression
of exogenous cellulose synthase genes in cyanobacteria which result
in the production and extracellular production of glucose.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application claims priority to and is a Divisional
Application of U.S. patent application Ser. No. 11/866,872, filed
Oct. 3, 2007 and U.S. Provisional Patent Application Ser. No.
60/849,363, filed Oct. 4, 2006, the entire contents of each of
which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Without limiting the scope of the invention, its background
is described in connection with cellulose production.
[0004] Cellulose biosynthesis has a significant impact on the
environment and human economy. The photosynthetic conversion of
CO.sub.2 to biomass is primarily accomplished through the creation
of the cellulosic cell walls of plants and algae (Lynd et al.,
2002). With approximately 10.sup.11 tons of cellulose created and
destroyed annually (Hess et al., 1928), this process ameliorates
the adverse effects of increased production of greenhouse gasses by
acting as a sink for CO.sub.2 (Brown, 2004). Although cellulose is
synthesized by bacteria, protists, and many algae; the vast
majority of commercial cellulose is harvested from plants.
[0005] Timber and cotton are the primary sources of raw cellulose
for a number of diverse applications including textiles, paper,
construction materials, and cardboard, as well as cellulose derived
products such as rayon, cellophane, coatings, laminates, and
optical films. Wood pulp from timber is the most important source
of cellulose for paper and cardboard. However, extensive processing
is necessary to separate cellulose from other cell wall
constituents (Klemm et al. 2005; Brown, 2004). Both the chemicals
utilized to extract cellulose from associated lignin and
hemicelluloses from wood pulp and the waste products generated by
this process pose serious environmental risks and disposal problems
(Bajpai, 2004). Additionally, the cultivation of other cellulose
sources, such as cotton, entails the extensive use of large tracts
of arable land, fertilizers and pesticides (both of which require
petroleum for their manufacture), and dwindling fresh water
supplies for irrigation.
SUMMARY OF THE INVENTION
[0006] More particularly, the present invention includes
compositions, methods, systems and kits for the production of
microbial cellulose using cyanobacteria that include a portion of
an exogenous cellulose operon sufficient to express bacterial
cellulose. Examples of cyanobacteria for use with the present
invention include those that are photosynthetic, nitrogen-fixing,
capable of growing in brine, facultative heterotrophs,
chemoautotrophic, and combinations thereof.
[0007] In one embodiment, the present invention includes
compositions and methods for isolated cyanobacteria that include a
portion of an exogenous bacterial cellulose operon sufficient to
express bacterial cellulose, whereby the cyanobacterium is capable
of the extracellular production of glucose. In one aspect, the
cyanobacterium is further defined as producing extracellular
glucose in the form of monosaccharides, disaccharides,
oligosaccharides or polysaccharides from photosynthesis. In another
aspect, the cyanobacterium is further defined as making
monosaccharides, disaccharides, oligosaccharides or polysaccharides
that comprise glucose and cellulose. Examples of cyanobacteria for
use with the present invention include Synechococcus sp. PCC 7002,
Synechococcus leopoliensis strain UTCC100, Agmenellum
quadruplicatum UTEX B2268, and Synechococcus sp. ATCC 27264.
Furthermore, the glucose, the cyanobacterial extracellular sheath
or both are further processed as a renewable feedstock for biofuel
production. In one aspect, the cyanobacterium can fix CO.sub.2
while producing cellulose and reducing atmospheric CO.sub.2 that
are quantified as carbon credits which are then sold in the open
market, e.g., a carbon credit market. In one aspect, the
cyanobacteria increase the extracellular production of
monosaccharides, disaccharides, oligosaccharides or polysaccharides
upon exposure to acidic conditions.
[0008] Another embodiment of the present invention includes an
isolated cyanobacterium, which includes a Synechococcus sp., with a
portion of an exogenous bacterial cellulose operon sufficient to
express bacterial cellulose, whereby the cyanobacterium is capable
of secreting monosaccharides, disaccharides, oligosaccharides or
polysaccharides that include glucose. In one aspect, the
cyanobacterium is further defined as producing extracellular
glucose in the form of monosaccharides, disaccharides,
oligosaccharides or polysaccharides from photosynthesis. In another
aspect, the cyanobacterium is further defined as making
monosaccharides, disaccharides, oligosaccharides or polysaccharides
that comprise glucose and cellulose. Example of cyanobacteria
include Synechococcus sp. PCC 7002, Synechococcus leopoliensis
strain UTCC100, Agmenellum quadruplicatum UTEX B2268, and
Synechococcus sp. ATCC 27264. The cellulose, the cyanobacterial
extracellular sheath or both are further processed as a renewable
feedstock for biofuel production.
[0009] Another method of the present invention includes producing a
photobiomass that may include monosaccharides, disaccharides,
oligosaccharides or polysaccharides that include glucose, by
modifying a cyanobacterium with a portion of an exogenous bacterial
cellulose operon sufficient to express and produce extracellular
glucose; growing the cyanobacterium under conditions that promote
extracellular glucose production; and exposing the cyanobacteria to
an acidic condition, wherein the acid increases glucose secretion.
The method may further include the step of processing the glucose
into ethanol. For example, the glucose is used as a renewable
feedstock for biofuel production, to fix CO.sub.2 and thus
atmospheric CO.sub.2 or even as a renewable feedstock for
animals.
[0010] Another embodiment of the present invention includes a
method of fixing carbon by growing a cyanobacterium comprising a
portion of an exogenous bacterial cellulose operon sufficient to
make cellulose and produce extracellular glucose in a
CO.sub.2-containing growth medium; generating glucose with said
cyanobacterium, wherein CO.sub.2 is fixed into glucose at a level
higher than an unmodified cyanobacterium; and calculating the
amount of CO.sub.2 fixed into the glucose to equate to one or more
carbon credit units. For example, at least one other carbon is
fixed into glucose and the at least one other carbon's is equated
to carbon credit units that is included in the calculation.
[0011] In another embodiment of the present invention includes an
isolated cyanobacterium that expresses a portion of an exogenous
bacterial cellulose operon sufficient to express bacterial
cellulose, whereby the cyanobacterium is capable of producing
extracellular monosaccharides, disaccharides, oligosaccharides or
polysaccharides.
[0012] A vector for expression of a portion of the cellulose operon
sufficient to express bacterial cellulose operon that includes a
microbial cellulose operon, e.g., the acsAB gene operon, under the
control of a promoter that expresses the genes in the operon in
cyanobacteria. The skilled artisan will recognize that the vector
may combine portions of the operons of bacterial, algal, fungal and
plant cellulose operons to maximize production and/or change the
characteristics of the cellulose and may be transfer and/or
expression vector.
[0013] The compositions and methods of the present invention also
include the use of the cyanobacteria-produced cellulose, which has
a lower crystallinity than wild-type bacterial cellulose and allows
for easier degradation to glucose for use in subsequent
fermentation to ethanol. One distinct advantage of the present
invention is that it permits very large scale production of
cellulose in areas that would otherwise not be available for
cellulose production (e.g., areas with little or no rainfall) while
at the same time producing cellulose with less toxic byproducts.
The cellulose of the present invention has a lower crystallinity
than wild-type bacterial cellulose and the lower crystallinity
cellulose is degraded with less energy into glucose than wild-type
cellulose and is further converted into ethanol.
[0014] The system for the manufacture of bacterial cellulose may
further include growing an exogenous cellulose expressing
cyanobacterium adapted for growth in a hypersaline environment,
such that the cyanobacterium does not grow in fresh water or the
salinity of sea water. The growth of the cyanobacteria in a
hypersaline environment may be used as way to limit the potential
for unplanned growth of the cyanobacteria outside controlled areas.
In one example, the cellulose expressing cyanobacteria of the
present invention may be grown in brine ponds obtained from
subterranean formation, such a gas and oil fields. Examples of
cyanobacteria for use with the system include those that are
photosynthetic, nitrogen-fixing, capable of growing in brine,
facultative heterotrophs, chemoautotrophic, and combinations
thereof. As with the previous embodiments of the present invention,
the cellulose genes may even obtained from mosses such as
Physcomitriella, algae, ferns, vascular plants, tunicates,
gymnosperms, angiosperms, cotton, switchgrass and combinations
thereof. The skilled artisan will recognize that it is possible to
combine portions of the operons of bacterial with algal, fungal and
plant cellulose genes to maximize production and/or change the
characteristics of the cellulose.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] For a more complete understanding of the features and
advantages of the present invention, reference is now made to the
detailed description of the invention along with the accompanying
figures and in which:
[0016] FIG. 1 shows a diagram of a production plant that may be
used to produce, isolate and process the saccharides produced using
the present invention.
[0017] FIG. 2 shows photobioreactor design for in situ harvest of
cyanobacterial saccharides.
[0018] FIG. 3 is a side view of a photobioreactor complex design
for in situ harvest of cyanobacterial saccharides.
DETAILED DESCRIPTION OF THE INVENTION
[0019] While the making and using of various embodiments of the
present invention are discussed in detail below, it should be
appreciated that the present invention provides many applicable
inventive concepts that can be embodied in a wide variety of
specific contexts. The specific embodiments discussed herein are
merely illustrative of specific ways to make and use the invention
and do not delimit the scope of the invention.
[0020] To facilitate the understanding of this invention, a number
of terms are defined below. Terms defined herein have meanings as
commonly understood by a person of ordinary skill in the areas
relevant to the present invention. Terms such as "a", "an" and
"the" are not intended to refer to only a singular entity, but
include the general class of which a specific example may be used
for illustration. The terminology herein is used to describe
specific embodiments of the invention, but their usage does not
delimit the invention, except as outlined in the claims.
[0021] As used herein the term, "cellulose" and "cellulose
substrate" include not only bacterial cellulose, but also native
cellulose from any source such trees, cotton, any vascular plant
(angiosperms and gymnosperms), any non-vascular plant such as
algae, mosses, liverworts, any animal that synthesizes cellulose
(such as tunicates or sea squirts), any prokaryotic organism (such
as cyanobacteria, purple bacteria, archaebacteria, etc. A complete
list and classification is available from the present inventors at:
http://128.83.195.51/cen/library/tree/cel.htm. As the inventors'
list shows, the cellulose may be from an organism that has one or
more cellulose synthase genes present. Furthermore, cellulose also
includes any derivatized form of cellulose such as cellulose
nitrate, acetate, carboxymethylcellulose, etc. Cellulose also
includes any form of native crystalline cellulose, which includes
not only the native crystalline form (called cellulose I, in its
alpha and beta sub allomorphs, all ratios, whether pure alpha or
pure beta). Cellulose for use with the present invention also
includes all processed crystalline celluloses, which deviates from
the native form of cellulose I, such as cellulose II (which is a
precipitated crystalline allomorph that is thermodynamically more
stable than cellulose I). Cellulose includes all variations of
molecular weights ranging from the lowest (oligosaccharides, 2-50
glucan monomers with a B-1,4 linkage), low molecular weight
celluloses with a degree of polymerization (dp), which is the
number of glucose molecules in the chain, of 50 to several hundred,
on up to the highest dp celluloses known (e.g., 15,000 from some
Acetobacter strains, to 25,000 from some algae). The present
invention may also use all variations of non crystalline cellulose,
including but not limited to, nematic ordered cellulose (NOC).
[0022] As used herein, the terms "continuous method" or "continuous
feed method" refer to a fermentation method that includes
continuous nutrient feed, substrate feed, cell production in the
bioreactor, cell removal (or purge) from the bioreactor, and
product removal. Such continuous feeds, removals or cell production
may occur in the same or in different streams. A continuous process
results in the achievement of a steady state within the bioreactor.
As used herein, the term "steady state" refers to a system and
process in which all of these measurable variables (i.e., feed
rates, substrate and nutrient concentrations maintained in the
bioreactor, cell concentration in the bioreactor and cell removal
from the bioreactor, product removal from the bioreactor, as well
as conditional variables such as temperatures and pressures) are
relatively constant over time.
[0023] As used herein, the terms "photobioreactor," "photoreactor,"
or "cyanobioreactor," include a fermentation device in the form of
ponds, trenches, pools, grids, dishes or other vessels whether
natural or man-made suitable for inoculating the cyanobacteria of
the present invention and providing to one or more of the
following: sunlight, artificial light, salt, water, CO.sub.2,
H.sub.2O, growth media, stirring and/or pumps, gravity or force fed
movement of the growth media. The product of the photobioreactor
will be referred to herein as the "photobiomass". The
"photobiomass" includes the cyanobacteria, secreted materials and
mass formed into, e.g., cellulose or value added products whether
intra or extracellular.
[0024] As used herein, the terms "bioreactor," "reactor," or
"fermentation bioreactor," include a fermentation device that
includes of one or more vessels and/or towers or piping
arrangement, which includes the Continuous Stirred Tank Reactor
(CSTR), Immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR),
Bubble Column, Gas lift Fermenter, Static Mixer, or other device
suitable for gas-liquid contact. A fermentation bioreactor for use
with the present invention includes a growth reactor which feeds
the fermentation broth to a second fermentation bioreactor, in
which most products, e.g., alkanols or furans are produced. In some
cases, the gaseous byproduct of fermentation, e.g., CO.sub.2, can
be pumped back into the photobioreactor to recycle the gas and
promote the formation of saccharides by photosynthesis. To the
extent that heat is generated during the process of recovering the
products of the fermentation, etc., the heat can also be used to
promote cyanobacterial cell growth and production of
saccharides.
[0025] As used herein, the term "nutrient medium" refers to
conventional cyanobacterial growth media that includes sufficient
vitamins, minerals and carbon sources to permit growth and/or
photosynthesis of the cellulose producing cyanobacteria of the
present invention. Components of a variety of nutrient media
suitable to the use of this invention are known and reported in
e.g., Cyanobacteria, Volume 167: (Methods in Enzymology)
(Hardcover), by John N. Abelson Melvin I. Simon and Alexander N.
Glazer (Editors), Academic Press, New York (1988).
[0026] As used herein, the term "cell concentration" refers to the
dry weight of cyanobacteria per liter of sample. Cell concentration
is measured directly or by calibration to a correlation with
optical density.
[0027] As used herein, the term "saccharide production" refers to
the amount of mono-, di-, oligo or polysaccharides produced by the
modified-cyanobacteria of the present invention that produce
saccharides by fixing carbon such as CO.sub.2 by photosynthesis
into the saccharides. One distinct advantage of the present
invention is that the cyanobacteria do not produce lignin along
with the production of the cellulose and other saccharides that can
be used as a feed-stock for fermentation and other bioreactors that
convert the saccharides into, e.g., ethanol or other synfuels.
[0028] In operation, the present invention may use any of a variety
of known fermentation process steps, compositions and methods for
converting the saccharides into useful products, e.g., lignin-free
cellulose, alkanols (alkyl alcohols), furans and the like. One
non-limiting example of a process for producing ethanol by
fermentation is a process that permits the simultaneous
saccharification and fermentation step by placing the saccharide
source at a temperature of above 34.degree. C. in the presence of a
glucoamylase and a thermo-tolerant yeast.
[0029] In this example, the following main process stages may be
included saccharification (if necessary), fermentation and
distillation. One particular advantage of the present invention is
that it eliminates a variety of processing steps, including,
milling, bulk-fiber separations, recovery or treatments for the
control or elimination of lignin, water removal, distillation and
burning of unwanted byproducts. Any of the process steps of alcohol
production may be performed batchwise, as part of a continuous flow
process or combinations thereof.
[0030] Saccharification. To produce mono- and di-saccharides from
the lignin-free cellulose of the present invention the cellulose
can be metabolized by cellulases that provide the yeast with simple
saccharides. This "saccharification" step include the chemical or
enzymatic hydrolysis of long-chain oligo and polysaccharides by
enzymes such as cellulase, glucoamylases, alpha-glucosidase,
alkaline, acid and/or thermophilic alpha-amylases and if necessary
phytases.
[0031] Depending on the length of the polysaccharides, enzymatic
activity, amount of enzyme and the conditions for saccharification,
this step may last up to 72 hours. Depending on the feedstock, the
skilled artisan will recognize that saccharification and
fermentation may be combined in a simultaneous saccharification and
fermentation step.
[0032] Fermentation. Any of a wide-variety of known microorganism
may be used for the fermentation, fungal or bacterial. For example,
yeast may be added to the feedstock and the fermentation is ongoing
until the desired amount of ethanol is produced; this may, e.g., be
for 24-96 hours, such as 35-60 hours. The temperature and pH during
fermentation is at a temperature and pH suitable for the
microorganism in question, such as, e.g., in the range about
32-38.degree. C., e.g. about 34.degree. C., above 34.degree. C., at
least 34.5.degree. C., or even at least 35.degree. C., and at a pH
in the range of, e.g., about pH 3-6, or even about pH 4-5. The
skilled artisan will recognize that certain buffers may be added to
the fermentation reaction to control the pH and that the pH will
vary over time.
[0033] The use of a feed stock that includes monosaccharides, in
addition to the use of thermostable acid alpha-amylases or a
thermostable maltogenic acid alpha-amylases and invertases in the
saccharification step may make it possible to improve the
fermentation step. When using a feedstock that includes large
amounts of monosaccharides such as glucose and sucrose, for the
production of ethanol it may be possible to reduce or eliminate the
need for the addition of glucoamylases in the fermentation step or
prior to the fermentation step.
[0034] Distillation. To complete the manufacture of final products
from the saccharides made by the cyanobacterial fixation of
CO.sub.2 of the present invention, the invention may also include
recovering the alcohol (e.g., ethanol). In this step, the alcohol
may be separated from the fermented material and purified with a
purity of up to e.g. about 96 vol. % ethanol can be obtained by the
process of the invention.
[0035] Several specific enzymes and methods may be used to improve
the recovery of energy containing molecules from the present
invention. The enzymes improve the saccharification and
fermentation steps by selecting their most efficient activity as
part of the processing of the products of the saccharide producing
modified cyanobacteria of the present invention.
[0036] In one example, a thermo tolerant cellulase may be
introduced into the reactor to convert cellulose produced by the
cyanobacteria of the present invention into monosaccharides, which
will mostly be glucose. Examples of thermophilic cellulases are
known in the art as taught in, e.g., U.S. Patent Application No
20030104522 filed by Ding, et al. that teach a thermal tolerant
cellulase from Acidothermus cellulolyticus. Yet another example is
taught by U.S. Patent Application No. 20020102699, filed by Wicher,
et al., which teaches variant thermostable cellulases, nucleic
acids encoding the variants and methods for producing the variants
obtained from Rhodothermus marinus. The relevant portions of each
are incorporated herein by reference.
[0037] Acid cellulase may be obtained commercially from
manufacturers such as Ideal Chemical Supply Company, Memphis Term.,
USA; Americos Industries Inc., Gujarat, India; Rakuto Kasei House,
Yokneam, Israel; or Novozymes, Bagsvaerd, Denmark. For example, the
acid cellulase may be provided in dry, liquid or high-active
abrasive form, as is commonly used in the denim acid washing
industry using techniques known to the skilled artisan. For
example, Americos Cellscos 450 AP is a highly concentrated acid
cellulase enzyme produced using genetically modified strains of
Trichoderma reesii. Typically, the acid cellulases function in a pH
range or 4.5-5.5.
[0038] Microorganisms used for fermentation. One example of a
microorganism for use with the present invention is a
thermo-tolerant yeast, e.g., a yeast that when fermenting at
35.degree. C. maintains at least 90% of the ethanol yields and 90%
of the ethanol productivity during the first 70 hours of
fermentation, as compared to when fermenting at 32.degree. C. under
otherwise similar conditions. One example of thermotolerant yeast
is a yeast that is capable of producing at least 15% V/V alcohol
from a corn mash comprising 34.5% (w/v) solids at 35.degree. C. One
such thermo-tolerant yeast is Red Star.RTM./Lesaffre Ethanol Red
(commercially available from Red Star.RTM./Lesaffre, USA, Product
No. 42138). The ethanol obtained using any known method for
fermenting saccharides (mono, di-, oligo or poly) may be used as,
e.g., fuel ethanol, drinking ethanol, potable neutral spirits,
industrial ethanol or even fuel additives.
[0039] Examples of ethanol fermentation from sugars are well-known
in the art as taught by, e.g., U.S. Pat. No. 4,224,410 to
Pemberton, et al. for a method for ethanol fermentation in which
fermentation of glucose and simultaneous-saccharification
fermentation of cellulose using cellulose and a yeast are improved
by utilization of the yeast Candida brassicae, ATCC 32196; U.S.
Pat. No. 4,310,629 to Muller, et al., that teaches a continuous
fermentation process for producing ethanol in which continuous
fermentation of sugar to ethanol in a series of fermentation
vessels featuring yeast recycle which is independent of the
conditions of fermentation occurring in each vessel is taught; U.S.
Pat. No. 4,560,659 to Asturias for ethanol production from
fermentation of sugar cane that uses a process for fermentation of
sucrose wherein sucrose is extracted from sugar cane, and subjected
to stoichiometric conversion into ethanol by yeast; and U.S. Pat.
No. 4,840,902 to Lawford for a continuous process for ethanol
production by bacterial fermentation using pH control in which a
continuous process for the production of ethanol by fermentation of
Zymomonas spp. is provided. The method of Lawford is carried out by
cultivating the organism under substantially steady state,
anaerobic conditions and under conditions in which ethanol
production is substantially uncoupled from cell growth by
controlling pH in the fermentation medium between a pH of about 3.8
and a pH less than 4.5; and K A Jacques, T P Lyons & D R
Kelsall (Eds) (2003), The Alcohol Textbook; 4.sup.TH Edition,
Nottingham Press; 2003. The relevant portions of each of which are
incorporated herein by reference.
[0040] One of ordinary skill in the art would recognize that the
quantity of yeast to be contacted with the photobiomass will depend
on the quantity of the photobiomass, the secreted portions of the
photobiomass and the rate of fermentation desired. The yeasts used
are typically brewers' yeasts. Examples of yeast capable of
fermenting the photobiomass include, but are not limited to,
Saccharomyces cerevisiae and Saccharomyces uvarum. Besides yeast,
genetically altered bacteria know to those of skill in the art to
be useful for fermentation can also be used. The fermenting of the
phototbiomass is conducted under standard fermenting
conditions.
[0041] Separating of the ethanol from the fermentation can be
achieved by any known method (e.g. distillation). The separation
can be performed on either or both the liquid and solid portions of
the fermentation solution (e.g., distilling the solid and liquid
portions), or the separation can just be performed on the liquid
portion of the fermentation solution (e.g., the solid portion is
removed prior to distillation). Ethanol isolation can be performed
by a batch or continuous process. The separated ethanol, which will
generally not be fuel-grade, can be concentrated to fuel grade
(e.g., at least 95% ethanol by volume) via additional distillation
or other methods known to those of skill in the art (e.g., a second
distillation).
[0042] The level of ethanol present in the fermentation solution
can negatively affect the yeast/bacteria. For example, if 17% by
volume or more ethanol is present, then it will likely begin
causing the yeast/bacteria to die. As such, ethanol can be
separated from the fermentation solution as the ethanol levels
(e.g., 12, 13, 14, 15, 16, to 17% by volume (ethanol to water))
that may kill the yeast or bacteria are reached. Ethanol levels can
be determined using methods known to those of ordinary skill in the
art.
[0043] The fermentation reaction can be run multiple times on the
photobiomass or portions thereof. For example, once the level of
ethanol in the initial fermentation reactor reaches 12-17% by
volume, the entire liquid portion of the fermentation solution can
be separated from the biomass to isolate the ethanol (e.g.,
distillation). The "once-fermented" photobiomass can then be
contacted with water, additional enzymes and yeast/bacteria for
additional fermentations, until the yield of ethanol is undesirably
low. Factors that the skilled artisan will use to determine the
number of fermentations include: the amount of photobiomass
remaining in the vessel; the amount of carbohydrate remaining, the
type of yeast or bacteria, the temperature, pH, salt concentration
of the media and overall ethanol yield. If any carbohydrates
remain, then the remaining photobiomass is removed from the
vessel.
[0044] Generally, it is desirable to isolate or harvest the
yeast/bacteria from the fermentation reaction for recycling. The
method of harvesting will depend upon the type of yeast/bacteria.
If the yeast/bacteria are top-fermenting, they can be skimmed off
the fermentation solution. If the yeast/bacteria are
bottom-fermenting, they can be removed from the bottom of the
tank.
[0045] Often, a by-product of fermentation is carbon dioxide, which
is readily recycled into the photobioreactor for fixation into
additional saccharides. During the fermentation process, it is
expected that about one-half of the decomposed starch will be
discharged as carbon dioxide. This carbon dioxide can be collected
by methods known to those of skill in the art (e.g., a floating
roof type gas holder) and is supplied back into the photobioreactor
pool or pools. In colder climates, the heat that may accompany the
carbon dioxide will help in the growth of the cyanobacterial
pools.
[0046] One advantage of the present invention is that it provides a
novel CO.sub.2 fixation method for the recycling of environmental
greenhouse gases. The present invention provides a source of
substrate for cellulose production from carbon dioxide that is
fixed into sugar by photosynthesis, thereby removing a major
barrier limiting large global scale production of cellulose. If the
present invention is successful on a large scale, it will sequester
CO.sub.2 from the air, thus reducing the potential greenhouse gas
responsible for global warming. Another benefit of the present
invention is that forests and cotton crops, the present sources for
cellulose, may not be needed in the future, thus freeing the land
to allow regeneration of forests and use of cropland for other
needs.
[0047] Microbial cellulose stands as a promising possible
alternative to traditional plant sources. The a proteobacterium
Acetobacter xylinum (synonym Gluconacetobacter xylinum [Yamada et
al., 1997]) is the most prolific of the cellulose producing
microbes. The NQ5 strain (Brown and Lin, 1990) is capable of
converting 50% of glucose supplied in the medium into an
extracellular cellulosic pellicle (R. Malcolm Brown, Jr., personal
communication). Although it possesses the same molecular formula as
cellulose derived from plant sources, microbial cellulose has a
number of distinctive properties that make it attractive for
diverse applications. The cellulose synthesized by A. xylinum is
"spun" into the growth medium as highly crystalline ribbons with
exceptional purity, free from the contaminating polysaccharides and
lignin found in most plant cell walls (Brown et al., 1976). The
resulting membrane or pellicle is composed of cellulose with a high
degree of polymerization (2000-8000) and crystallinity (60-90%)
(Klemm, et al., 2005). Contaminating cells are easily removed, and
relatively little processing is required to prepare membranes for
use. In its never-dried state, the membrane displays exceptional
strength and is highly absorbent, holding hundreds of times its
weight in water (White and Brown, 1989). A. xylinum cellulose is
therefore, well suited as a reinforcing agent for paper and diverse
specialty products (Shah and Brown, 2005; Czaja et al., 2006;
Tabuchi et al., 2005; Helenius et al., 2006).
[0048] The acsAB genes from the cellulose synthase operon of or the
gram negative bacterium, Acetobacter xylinum (=Gluconacetobacter
xylinus) under control of a lac promoter have been integrated into
the chromosome of a photosynthetic cyanobacterium, Synechococcus
leopoliensis. UTCC 100. The presence of the genes in the chromosome
has been confirmed by PCR. Preliminary data from Western analysis,
light microscopy, and growth characteristics suggests functional
expression of these genes in Synechococcus. Cyanobacteria
expressing exogenous cellulose synthase genes will be used for the
efficient and inexpensive production of bacterial cellulose.
[0049] Despite it superior quality, the use of microbial cellulose
as a primary constituent for large scale use in common applications
such as the production of construction materials, paper, or
cardboard has not been economically feasible. The root cause for
the expense of microbial cellulose production is the heterotrophic
nature of A. xylinum. Bacterial cultures must be supplied with
glucose, sucrose, fructose, glycerol, or other carbon sources
produced by the cultivation of plants. Increased distance from the
primary energy source is inherently less efficient and inevitably
leads to increased cost of production when compared with
phototrophic sources. Therefore, while the unique properties of A.
xylinum cellulose make it indispensable for a number of value added
products, it is not well suited for the more general applications
that constitute the vast majority of cellulose utilization (Brown,
2004; White and Brown, 1989), e.g., to replace the use of forests
for the production of paper and to provide substrates for the
production of biofuels based on ethanol using photosynthesis as the
source of energy for CO.sub.2 fixation. As such, the present
invention provides compositions and methods for the manufacture of
a new global crop that may be used for energy production and
removal of the greenhouse gas CO.sub.2 using an environmentally
acceptable natural process that requires little or no energy input
for manufacture.
[0050] Currently, bacterial cellulose is produced by A. xylinum, a
heterotrophic a proteobacterium. The fact that the precursor of
cellulose, namely glucose, needs to be supplied, presents a
bottleneck in large scale production of microbial cellulose.
Present technology would suggest using sugarcane extracts, sucrose,
beet sugar, etc., as sources. If the rate of cellulose biosynthesis
in cyanobacteria is increased via the expression of exogenous
cellulose synthase genes, then the potential for an economical
global cellulose crop is possible. Cellulose synthase genes have
been stably integrated into the chromosome by recombination but
also could be expressed on replicating plasmids.
[0051] Unlike A. xylinum, cyanobacteria require no fixed carbon
source for growth. Additionally, many cyanobacteria are capable of
nitrogen fixation, which would eliminate the need for fertilizers
necessary for cellulose crops like cotton. Furthermore, many
cyanobacteria are halophilic, that is, they can grow in a the range
of brackish to hypersaline environments. This feature, in
combination with N-fixation, will allow non-arable, sun-drenched
areas of the planet to provide the extensive surface areas for the
growth and harvest of cellulose made using the compositions and
methods of the present invention on a global scale.
[0052] Cyanobacterial cellulose can be used in diverse applications
where a combination of products is simultaneously made from
photosynthesis. Value added products may include pharmaceuticals
and/or vaccines, vitamins, industrial chemicals, proteins,
pigments, fatty acids and their derivatives (such as
polyhydroxybutyrate), acylglycerols (as precursors for biodiesel),
as well as other secondary metabolites. These products may be the
result of natural cyanobacterial metabolic processes or be induced
through genetic engineering. The present invention permits large
scale production of cellulose, proteins and other products that may
be grown and harvested. In fact, wide application of the cells
themselves for glucose and cellulose is encompassed by the present
invention. The cellulose producing cyanobacteria of the present
invention may be utilized for energy recycling and recovery, that
is, the cells may be dried and burned to power downstream processes
in a manner similar to the use of bagasse in the sugar cane
industries.
[0053] The ideal cellulose producing organism would synthesize
cellulose of a quality and in the quantities observed in A.
xylinum, have a photoautotrophic lifestyle, and possess the ability
to grow with a minimum use of natural resources in environments
unsuitable for agriculture. Cyanobacteria are capable of using low
photon flux densities for carbon fixation, withstanding hypersaline
environments, tolerating desiccation, and surviving high levels of
UV irradiation (Vincent, 2000; Wynn-Williams, 2000). Additionally,
many species are diazotrophic (Castenholz and Waterbury, 1989).
This combination of exceptional adaptive characteristics has made
mass cultivation of cyanobacteria attractive for production of
nutritional biomass, fatty acids, bioactive compounds, and
polysaccharides (Cogne et al., 2005; Moreno et al., 2003; Kim et
al., 2005). Although no species of cyanobacteria are known to
synthesize cellulose in large quantities, the development of a
number of systems for engineering of cyanobacterial chromosomes may
offer a means to a new global crop of cellulose produced by
cyanobacteria.
[0054] Toward this end, genes that include the cellulose synthase
operon of A. xylinum NQ5 were integrated into the chromosome of the
unicellular cyanobacterium, Synechococcus leopoliensis UTCC 100
(synonym Synechococcus elongatus PCC 7942). Alternatively, a
cyanobacterium for use with the present invention may be a
salt-water variety that is diazotrophic. S. elongatus has served as
a model organism for molecular studies of photosynthesis and
circadian rhythms, and has been successfully utilized for
transgenic expression (Rixin and Golden, 1993; Nair et al., 2000;
Deng and Coleman, 1999; Asada et al., 2000). S. elongatus has a
rapid growth rate, readily recombines DNA into its chromosome by
transformation or conjugation, can act as a host for replicating
plasmids, and its physiology, genetics, and biochemistry are well
characterized (Golden et al., 1987; Thiel, 1995; Deng and Coleman,
1999). Additionally, a project to sequence the genome of this
organism is underway
(<genome.jgi-psf.org/finished_microbes/synel/synel.home.html&-
gt;). These characteristics facilitate the transfer and expression
of exogenous genes and manipulation of native regulatory
components.
[0055] Culture Conditions. Genetically modified strains of
Synechococcus (see Table I for a description of strains) were
maintained at 24.degree. C. with 12 hour light/dark cycles using
BG11 (Allen, 1968) as the growth medium. Solid media was prepared
with 1.5% agar as previously described (Golden, 1988). 50 ml liquid
cultures were maintained on a rotary shaker in 250 ml Erlenmeyer
flasks. Growth media was supplemented with 7.5 ug/ml
chloramphenicol. Cell concentrations of cultures were determined by
measuring their optical density at 750 nm (OD.sub.750).
TABLE-US-00001 TABLE 1 Strain Characteristics. Strain Relevant
Characteristics NS::cat Synechococcus leopoliensis UTCC 100 strain
carrying the chloramphenicol acetyltransferase marker in
chromosomal neutral site II. This strain was created using vector
pAM1573. NS::ab.DELTA.c7s Synechococcus leopoliensis UTCC 100
strain carrying acsAB.DELTA.C from Gluconacetobacter xylinum strain
NQ5 and the chloramphenicol acetyltransferase marker in chromosomal
neutral site II. This strain was created using vector pSAB2.
[0056] Determination of Glucose Concentrations
[0057] Preparation of Cultures. 50 ml liquid cultures were
inoculated by scraping cells from the surface of agar plates with
flame-sterilized spatulas such that the initial OD.sub.750 was
1.67+/-0.22. Cultures of NS::cat and NS::ab.DELTA.c7S were grown
for 7-14 days under the conditions described above. The OD.sub.750
of each culture was recorded. Cells from 40 ml aliquots of liquid
cultures were collected by centrifugation (10 min, RT,
1,744.times.g) in an IEC clinical centrifuge. The supernatants were
discarded and wet weights of the cell pellets were recorded.
Pellets were resuspended in 1 ml of 10 mM Sodium Acetate, pH 5.2.
250 ul aliquots of the cell suspension were transferred to 1.5 ml
eppendorf tubes. The tubes were incubated overnight on a rotisserie
at 30.degree. C. with constant illumination.
[0058] Glucose Assays. After overnight incubation, cells were
pelleted by centrifugation (5 min, RT, 14,000 rpm) in a
microcentrifuge. The supernatant was carefully pipetted off the
cell pellet and retained for the glucose assay. Glucose
concentration was measured using the hexokinase, glucose
6-phosphate dehydrogenase enzymatic assay (Sigma G3293). Assays
were performed with 50-100 ul of supernatant per reaction following
the manufacturer's instructions.
[0059] Table 2 demonstrates that the expression of genes from the
cellulose synthase operon of Gluconacetobacter xylinus strain NQ5
in NS::ab.DELTA.c7S results in an order of magnitude increase in
the production of glucose when compared to NS::cat. Assuming
lossless scale-up, the observed extracellular glucose production
levels of NS::ab.DELTA.c7S would translate into approximately 380
gallons of ethanol per acre foot per year. This is comparable to
current production levels of corn (400 gallons of ethanol per acre)
and is roughly one third of the productivity of switchgrass (1150
gallons per acre per year). However, it is important to note that
the glucose being produced by our strain does not require extensive
pretreatment nor does it require the application of exogenous
cellulose digesting enzymes. Thus, the two most costly steps in the
conversion of biomass to ethanol are eliminated. Therefore, even
with lower production levels, cyanobacterial glucose may be an
economically feasible feedstock for ethanol production.
[0060] Table 2. Comparison of glucose production levels. Values
representing cell concentrations, cell mass, and glucose production
by NS::cat and NS::ab.DELTA.c7S. Optical densities and wet weights
were recorded prior to resuspension in 10 mM Sodium Acetate, pH
5.2. The glucose concentration in mg/ml was measured from aliquots
of cell suspensions resulting from the concentration of 40 ml of
liquid culture into 1 ml of Sodium Acetate.
TABLE-US-00002 TABLE 2 Comparison of glucose production levels. Wet
weight Glucose mg Glucose mg Glucose Strain OD.sub.750 (g) (mg/ml)
g wet weight liter NS::cat 1.65 +/- 0.13 0.35 +/- 0.10 0.12 +/-
0.06 0.17 +/- 0.25 1.03 +/- 1.40 NS::ab.DELTA.c7S 1.82 +/- 0.19
0.41 +/- 0.15 1.37 +/- 0.06 3.70 +/- 1.55 34.32 +/- 1.62
[0061] Not wanting to be bound by theory, several possible
mechanisms leading to the release of free glucose into the external
milieu may exist. Glucose may be exuded from cells or released from
extracellular polysaccharides by the actions of one or more
endogenous secreted glycosyl hydrolases, e.g., Syn_PCC79421400 (see
e.g.,
<maple.lsd.ornl.gov/cgi-bin/JGI_microbial/gene_viewer.cgi?org=syn_PCC7-
942&chr=21jun05&contig=Contig52&gene=Syn_pcc79421400>)
capable of acting on non-crystalline cellulosic material, Discovery
of the mechanism responsible for the observed glucose levels will
almost certainly uncover novel biological processes and may provide
the means for increased glucose production in this organism.
[0062] FIG. 1 shows one example of a photobioreactor system 100 of
the present invention. First, inputs 102 for the photobioreactor
system may include: sunlight, salt, water, CO.sub.2
modified-cyanobacterial cells of the present invention, growth
medium components and if necessary a source of power to move the
components (e.g., pumps or gravity). Next, the inputs 102 and
inoculated into a photobioreactor grid 104 that is used to grow the
modified-cyanobacteria in size and number, to test for saccharide
production and to reach a sufficiently high enough concentration to
inoculate the operating photobioreactor 106. The photobioreactor
106 may be a pool or pool(s), trench or other vessel, indoor or
outdoor that is used to grow and harvest a sufficient volume of
photobiomass for subsequent processing in, e.g., processing plant
110. In one example, the photobioreactor 106 may be a grid of pools
of one square mile (or larger) that may be used in parallel or in
series to produce the photobiomass. Depending on the geographical
location of the photobioreactor 106, the water may be saltwater or
brine obtained from a sea that is gravity fed into the pools.
Gravity or pumping may be used, however, gravity has the advantage
that it does not require additional energy to move the photobiomass
from pool to pool and even into the processing plant. In fact, in
certain embodiments the entire system may be gravity fed with the
final products gravity fed into underground rivers that return to
the sea or ocean.
[0063] The processing plant 110 includes a cell harvested 112,
which may allows the isolation of the photobiomass by, e.g.,
centrifugation, filtration, sedimentation, creaming or any other
method for separating the photobiomass, the modified-cyanobacterial
cells and the liquid. For the isolation of sucrose, the cells may
be resuspended in medium with an increased salinity 114 (e.g.,
2.times. the salinity) followed by a second harvesting step 116.
The twice-harvested cells are then resuspended under acidic
conditions (e.g., pH 4.5-5.5) at 40 to 100.times. the concentration
and the sucrose is secreted by the modified-cyanobacteria. If
glucose is preferred, the once harvested cells are resuspended
under acidic conditions 118 and glucose is secreted. In addition,
whether sucrose or glucose is secreted, cellulose is also harvested
from the modified-cyanobacteria, which may be further digested by
cellulases 120. Glucose and digested cellulose can then be
fermented into ethanol or other alkanols 122.
[0064] If sucrose is secreted and obtained, then the sucrose can be
converted into dimethylfuran and glucose by invertase 124. The
methylfuran 126 can then be used for bioplastic 130 or biofuel 128
production. Glucose that is obtained after the invertase reaction
124 can then be directed back into the fermentation reactions.
[0065] In addition to the production of ethanol, bioplastics and
other biofuels, the harvested cells can be used for the production
of other high value bioproducts 132, e.g., by further modifying the
microbial cellulose-producing cyanobacteria to make other
bioproducts, e.g., pharmaceuticals and/or vaccines, vitamins,
industrial chemicals, proteins, pigments, fatty acids and their
derivatives (such as polyhydroxybutyrate), acylglycerols (as
precursors for biodiesel), as well as other secondary metabolites.
After each of these steps, the modified-cyanobacteria can then be
recycled into the photobioreactors for additional carbon fixation
134. Furthermore, the products of the processing plant 110 can also
be combined with other power sources, e.g., solar, methane, wind,
etc., to generate electricity and heat 136 (in addition to
recycling any CO.sub.2 released in the processing plant 110), to
power the inoculation pool 104 and the photobioreactor 106.
[0066] FIG. 2 shows a photobioreactor design for in situ harvest of
cyanobacterial saccharides. The photobioreactor complex can be
located indoors or underground. Part A An LED array powered by
photovoltaic cells, provides mono or polychromatic light at a
pulsed frequencies corresponding to the rate limiting steps of
photosynthesis for maximized photosynthetic productivity Part B is
a transparent photobioreactor acting as a growth vessel for
cyanobacterial cells. The horizontal orientation of the
photobioreactor allows for efficient separation of cells from
culture medium by use of gravity and air pressure. Part C is a
filter screen combined with a water release trap will separate
cells from the culture medium. The filter screen will have pore
sizes capable of retaining cyanobacterial cells while allowing
culture medium to flow into the reservoir. The transfer will be
facilitated by gravity and air pressure generated by closing the
gas outlet of the photobioreactor. The reservoir, located beneath
the photobioreactor, will act to retain culture medium during
harvest of saccharides. After harvest, culture medium will be
returned to the photobioreactor from the reservoir via pump.
[0067] FIG. 3 shows the operation of a photobioreactor complex
design for in situ harvest of cyanobacterial saccharides. The LED
array, located on top of the photobioreactor complex will supply
pulsed mono or polychromatic light for maximum photosynthetic
conversion efficiency. Air flow (CO.sub.2, N.sub.2, or ambient air)
delivered by the gas inlet during growth periods will serve to
deliver carbon and/or nitrogen sources for fixation and created
turbulence for maintaining cell suspension. A gas outlet will
facilitate the release of waste gasses (O.sub.2 and H.sub.2) that
are potentially detrimental to the cyanobacterial growth and
relieve excess air pressure from the system during growth phases.
Removal of culture media for harvesting of saccharides will be
facilitated by the opening of the liquid release trap coupled with
closing the gas outlet. The increase in air pressure, combined with
gravity, will force the culture medium through the filter which
will retain cyanobacterial cells. Cyanobacterial cells can then be
resuspended in specific buffer or media designed for cellulose
digestion or the direct secretion of saccharides. The
saccharide-containing solutions will be drained to chamber 2 of the
liquid release trap by the same method described for growth media
above. Soluble saccharides will be pumped from chamber 2 of the
reservoir to central processing units for downstream conversion
processes (e.g., fermentation, chemical conversion to
dimethylfuran, etc.). Cells will be resuspended by closing the
water release trap and pumping culture medium which has been
recombined with fresh media components (e.g., nitrates, phosphates,
etc.) from chamber 1 of the reservoir.
[0068] It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method, kit,
reagent, or composition of the invention, and vice versa.
Furthermore, compositions of the invention can be used to achieve
methods of the invention.
[0069] It will be understood that particular embodiments described
herein are shown by way of illustration and not as limitations of
the invention. The principal features of this invention can be
employed in various embodiments without departing from the scope of
the invention. Those skilled in the art will recognize, or be able
to ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of this invention
and are covered by the claims.
[0070] All publications and patent applications mentioned in the
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
[0071] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one." The use of
the term "or" in the claims is used to mean "and/or" unless
explicitly indicated to refer to alternatives only or the
alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0072] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, unrecited elements or method steps.
[0073] The term "or combinations thereof" as used herein refers to
all permutations and combinations of the listed items preceding the
term. For example, "A, B, C, or combinations thereof" is intended
to include at least one of: A, B, C, AB, AC, BC, or ABC, and if
order is important in a particular context, also BA, CA, CB, CBA,
BCA, ACB, BAC, or CAB. Continuing with this example, expressly
included are combinations that contain repeats of one or more item
or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so
forth. The skilled artisan will understand that typically there is
no limit on the number of items or terms in any combination, unless
otherwise apparent from the context.
[0074] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended
claims. [0075] Allen M. (1968). Simple conditions for growth of
unicellular blue green algae on plates. J Phycol 4: 1-4. [0076]
Golden S S, Brusslan J, Haselkorn R. (1988). Mutagenesis of
cyanobacteria by classical and gene-transfer-based methods. In
Packer L and Glazer A N (eds) Methods in Enzymology ed. Vol. 167 pp
714-727. Academic Press, Inc. New York. [0077] Nobles D R,
Romanovicz D K, Brown R M Jr. (2001). Cellulose in cyanobacteria.
Origin of vascular plant cellulose synthase? Plant Physiol.
127(2):529-42. [0078] Roberts E. (1991). Biosynthesis of Cellulose
II and Related Carbohydrates PhD thesis. The University of Texas at
Austin, Austin. [0079] Roelofsen P A. (1959). The plant cell wall
constituents. In: The Plant Cell Wall. Gebruder Borntraeger (ed).
Felengraff and Co. Berlin. pp 1-33. [0080] Sakamoto T and Bryant D
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Characterization of genes in the cellulose synthesizing operon (acs
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[0083] Tel-Or E, Spath S, Packer L, and Mehlhorn R J. (1986).
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* * * * *
References