U.S. patent application number 15/859219 was filed with the patent office on 2018-12-27 for microalgae fermentation using controlled illumination.
The applicant listed for this patent is Phycoil Biotechnology International, Inc.. Invention is credited to Chung-Soon Im, Jane Kim.
Application Number | 20180371395 15/859219 |
Document ID | / |
Family ID | 43759033 |
Filed Date | 2018-12-27 |
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United States Patent
Application |
20180371395 |
Kind Code |
A9 |
Im; Chung-Soon ; et
al. |
December 27, 2018 |
MICROALGAE FERMENTATION USING CONTROLLED ILLUMINATION
Abstract
Bioreactors and methods for cultivating microalgae are provided
herein. The bioreactor and methods include features and
modifications to improve heterotrophic growth efficiency by
providing a light signal.
Inventors: |
Im; Chung-Soon; (Palo Alto,
CA) ; Kim; Jane; (San Jose, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Phycoil Biotechnology International, Inc. |
Menlo Park |
CA |
US |
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Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20180142197 A1 |
May 24, 2018 |
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Family ID: |
43759033 |
Appl. No.: |
15/859219 |
Filed: |
December 29, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13395107 |
Mar 8, 2012 |
9932554 |
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PCT/US10/49347 |
Sep 17, 2010 |
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15859219 |
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61359726 |
Jun 29, 2010 |
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61243593 |
Sep 18, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02T 50/678 20130101;
C12M 21/02 20130101; A01G 33/00 20130101; Y02E 50/13 20130101; C12N
1/12 20130101; Y02P 20/134 20151101; C12P 7/6463 20130101; C12M
21/12 20130101; Y02E 50/10 20130101; C12M 31/08 20130101; C12P
7/649 20130101; C12M 31/02 20130101; Y02P 20/133 20151101; C12M
31/10 20130101 |
International
Class: |
C12M 1/00 20060101
C12M001/00; C12N 1/12 20060101 C12N001/12; C12P 7/64 20060101
C12P007/64; A01G 33/00 20060101 A01G033/00 |
Claims
1. A method for cultivating a microalgae comprising a photoreceptor
sensitive to a particular region of a visible spectrum under a
heterotrophic growth condition, comprising: incubating the
microalgae under the heterotrophic growth condition for a period of
time sufficient to allow the microalgae to grow, wherein the
heterotrophic growth condition comprises: a media comprising a
carbon source, and an irradiance of light including the particular
region of the visible spectrum in a sufficient intensity to
transduce the photoreceptor.
2. The method of claim 1, wherein the microalgae is a Botryococcus
strain, wherein the carbon source is glucose, and wherein the
irradiance of light is between 1-10 .mu.mol photons
m.sup.-2s.sup.-1.
3. The method of claim 1, wherein the microalgae is a Botryococcus
sudeticus strain, a Botryococcus strain, a UTEX 2629 strain, a
Botryococcus braunii strain, a UTEX 2441 strain, a Neochloris
oleabundans strain, a Neochloris strain, a UTEX 1185 strain, a
Chlamydomonas reinhardtii strain, a Chlamydomonas strain, a UTEX
2243 strain, or a strain comprising a photoreceptor.
4. The method of claim 2, wherein the microalgae is a UTEX 2629
strain or a UTEX 2441 strain.
5. The method of claim 1, wherein the carbon source is glucose.
6. The method of claim 1, wherein the carbon source is selected
from the group consisting of a fixed carbon source, glucose,
fructose, sucrose, galactose, xylose, mannose, rhamnose,
N-acetylglucosamine, glycerol, floridoside, glucuronic acid, corn
starch, depolymerized cellulosic material, sugar cane, sugar beet,
lactose, milk whey, and molasses.
7. The method of claim 1, wherein the light is produced by an
artificial light source, or the light is artificial light.
8. The method of claim 1, wherein the intensity of the irradiance
of light is between 0.01-1 .mu.mol photons m.sup.-2s.sup.-1,
between 1-10 .mu.mol photons m.sup.-2s.sup.-1, between 10-100
.mu.mol photons m.sup.-2s.sup.-1, between 100-300 .mu.mol photons
m.sup.-2s.sup.-1, 3-4 .mu.mol/m.sup.2s.sup.-1 photons, 2-3
.mu.mol/m.sup.2s.sup.-1 photons, 1-2 .mu.mol/m.sup.2s.sup.-1
photons, or 3-5 .mu.mol/m.sup.2s.sup.-1 photons.
9. The method of claim 1, further comprising producing a material
from the microalgae.
10. The method of claim 9, wherein the material is a
polysaccharide, a pigment, a lipid, or a hydrocarbon.
11. The method of claim 9, wherein the material is a
hydrocarbon.
12. The method of claim 9, further comprising recovering the
material.
13. The method of claim 9, further comprising extracting the
material.
14. The method of claim 9, further comprising processing the
material.
15. The method of claim 12, further comprising processing the
material.
16. The method of claim 14, wherein the processing of the material
produces a processed material.
17. The method of claim 16, wherein the processed material is
selected from the group consisting of a fuel, biodiesel, jet fuel,
a cosmetic, a pharmaceutical agent, a surfactant, and a renewable
diesel.
18. A method of manufacturing a material, comprising: providing a
microalgae comprising a photoreceptor sensitive to a particular
region of a visible spectrum and capable of producing the material;
culturing the microalgae in a media, wherein the media comprises a
carbon source; applying an irradiance of light including the
particular region of the visible spectrum in a sufficient intensity
to transduce the photoreceptor; and allowing the microalgae to
accumulate at least 10% of its dry cell weight as the material.
19. A bioreactor system, comprising: a bioreactor; a culture medium
comprising a carbon source, wherein the culture medium is located
inside the bioreactor; a microalgae comprising a photoreceptor
sensitive to a particular region of a visible spectrum and adapted
for heterotrophic growth, wherein the microalgae is located in the
culture media; and a light source, wherein the light source
produces an irradiance of light including the particular region of
the visible spectrum in a sufficient intensity to transduce the
photoreceptor, and wherein the light source is operatively coupled
to the bioreactor.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/243,593, filed Sep. 18, 2009, and U.S.
Provisional Application No. 61/359,726, filed Jun. 29, 2010, the
entire disclosures of which are hereby incorporated by reference in
their entirety for all purposes.
FIELD
[0002] The invention relates to methods, means, and systems of
fermentation of microorganisms, e.g., microalgae. The invention can
be used in pharmaceutical, cosmetic and food industries, as well as
for obtaining oil and biofuel from microalgae.
BACKGROUND
[0003] Recently, attention has been directed to the application of
microalgae to the production of a variety of materials including
lipids, hydrocarbons, oil, polysaccharides, pigments, and
biofuels.
[0004] One of the conventional methods to grow microalgae is to
heterotrophically culture it in an enclosed, light-free system.
Techniques have been developed for the large-scale production of
aquatic microalgae under heterotrophic growth conditions by
utilizing organic carbon instead of light as an energy source. For
example, U.S. Pat. Nos. 3,142,135 and 3,882,635 describe processes
for the heterotrophic production of proteins and pigments from
algae such as Chlorella, Spongiococcum, and Prototheca. In
addition, heterotrophic algal cultures can attain much higher
densities than photoautotrophic cultures.
[0005] However, the above application cannot be applied to all
microalgae because only a limited number of microalgae strains can
grow in heterotrophic conditions. Attempts to grow microalgae in
heterotrophic conditions often involve either screening for strains
that can grow in heterotrophic conditions or genetic modification
of organisms to allow growth under such conditions.
[0006] Microalgae that contain both a proper transportation system
for sugar and that can grow naturally in heterotrophic conditions
often show slow growth rates or low production of materials of
commercial interest since they have evolved many years to utilize
sunlight as an environmental signal to control aspects of
metabolism as well as energy generated through photosynthesis.
[0007] Most of the photosynthetic organisms, including microalgae,
use light as an environmental signal to optimize themselves for
growth and survival. Light signals are sensed by different
photoreceptors including red/far-red photoreceptors (phytochromes)
and blue light photoreceptors (cryptochromes and NPHs). Light
serves as an environmental signal that regulates physiological and
developmental processes and provides the energy that fuels the
reduction of inorganic carbon. However, under certain conditions
light also has the potential to be toxic. Photoinhibition occurs
either when the photon flux absorbed by chloroplasts is very high
(under high light conditions) or when the light energy influx
exceeds the consumption capability (under mixotrophic conditions
where a cell uses reduced carbon as an energy source). In
mixotrophic conditions, photosynthetic organisms show
photoinhibition at a much lower light intensity than autotrophic
conditions since the electrons absorbed through the photosynthetic
apparatus cannot be efficiently used due to a feedback mechanism in
the Calvin cycle.
[0008] Absorbed light energy can result in the accumulation of
excited chlorophyll molecules within the pigment bed and damage of
the photosystem. Excited chlorophyll molecules that accumulate in
the pigment bed as a consequence of excess excitation can also
stimulate the production of active oxygen species such as
superoxides, hydroxyl radicals and singlet oxygen.
SUMMARY
[0009] Disclosed herein is a method for cultivating a microalgae
capable of heterotrophic growth, including: incubating the
microalgae under a heterotrophic growth condition for a period of
time sufficient to allow the microalgae to grow, wherein the
heterotrophic growth condition includes a media including a carbon
source, and wherein the heterotrophic growth condition further
includes a low irradiance of light.
[0010] In some embodiments, the microalgae is a Botryococcus
strain, the carbon source is glucose, and the low irradiance of
light is between 1-10 .mu.mol photons m.sup.2 s.sup.-1.
[0011] In some embodiments, the microalgae is a Botryococcus
sudeticus strain. In some embodiments, the microalgae is a
Botryococcus strain. In some embodiments, the microalgae is a UTEX
2629 strain. In some embodiments, the microalgae is a Botryococcus
braunii strain. In some embodiments, the microalgae is a UTEX 2441
strain. In some embodiments, the microalgae is a Neochloris
oleabundans strain. In some embodiments, the microalgae is a
Neochloris strain. In some embodiments, the microalgae is a UTEX
1185 strain. In some embodiments, the microalgae is a Chlamydomonas
reinhardtii strain. In some embodiments, the microalgae is a
Chlamydomonas strain. In some embodiments, the microalgae is a UTEX
2243 strain. In some embodiments, the microalgae comprises a
photoreceptor.
[0012] In some embodiments, the carbon source is glucose. In some
embodiments, the carbon source is selected from the group
consisting of a fixed carbon source, glucose, fructose, sucrose,
galactose, xylose, mannose, rhamnose, N-acetylglucosamine,
glycerol, floridoside, glucuronic acid, corn starch, depolymerized
cellulosic material, sugar cane, sugar beet, lactose, milk whey,
and molasses.
[0013] In some embodiments, the light is produced by a natural
light source. In some embodiments, the light is natural sun light.
In some embodiments, the light comprises full spectrum light or a
specific wavelength of light. In some embodiments, the light is
produced by an artificial light source. In some embodiments, the
light is artificial light. In some embodiments, the intensity of
the low irradiance of light is between 0.01-1 .mu.mol photons
m.sup.-2 s.sup.-1. In some embodiments, the intensity of the low
irradiance of light is between 1-10 .mu.mol photons m.sup.-2
s.sup.-1. In some embodiments, the intensity of the low irradiance
of light is between 10-100 .mu.mol photons m.sup.-2 s.sup.-1. In
some embodiments, the intensity of the low irradiance of light is
between 100-300 .mu.mol photons m.sup.-2 s.sup.-1. In some
embodiments, the intensity of the low irradiance of light is
between 100-300 .mu.mol photons m.sup.-2 s.sup.-1. In some
embodiments, the intensity of the low irradiance of light is 3-4
.mu.mol/m.sup.2s.sup.1 photons, 2-3 .mu.mol/m.sup.2s.sup.1 photons,
1-2 .mu.mol/m.sup.2s.sup.-1 photons, or 3-5 .mu.mol/m.sup.2s.sup.-1
photons.
[0014] In some embodiments, the method further includes producing a
material from the microalgae. In some embodiments, the material is
a polysaccharide, a pigment, a lipid, or a hydrocarbon. In some
embodiments, the material is a hydrocarbon.
[0015] In some embodiments, the method further includes recovering
the material. In some embodiments, the method further includes
extracting the material.
[0016] In some embodiments, the method further includes processing
the material. In some embodiments, the processing of the material
produces a processed material. In some embodiments, the processed
material is selected from the group consisting of a fuel,
biodiesel, jet fuel, a cosmetic, a pharmaceutical agent, a
surfactant, and a renewable diesel.
[0017] In some embodiments, the growth rate of the microalgae in
the above methods is higher than a second microalgae incubated
under a second heterotrophic growth condition for a period of time
sufficient to allow the microalgae to grow, wherein the second
heterotrophic growth condition includes a growth media comprising a
carbon source, and wherein the second heterotrophic growth
condition does not include a low irradiance of light.
[0018] Also described herein is a method of culturing microalgae,
including placing a plurality of microalgae cells in the presence
of a carbon source and a low irradiance of light.
[0019] Also described herein is a method of manufacturing a
material, including: providing a microalgae capable of producing
the material; culturing the microalgae in a media, wherein the
media includes a carbon source; applying a low irradiance of light
to the microalgae; and allowing the microalgae to accumulate at
least 10% of its dry cell weight as the material. In some
embodiments, the method further includes recovering the
material.
[0020] Also described herein is a bioreactor system, including: a
bioreactor; a culture media including a carbon source, wherein the
culture media is located inside the bioreactor; a microalgae
adapted for heterotrophic growth, wherein the microalgae is located
in the culture media; and a light source, wherein the light source
produces a low irradiance of light, and wherein the light source is
operatively coupled to the bioreactor.
[0021] In some embodiments, light from the light source includes
full spectrum light or a specific wavelength of light. In some
embodiments, light from the light source includes natural sunlight
collected by a solar energy collector operatively coupled to the
bioreactor, and wherein the light is transmitted to the interior of
the bioreactor through an optical fiber operatively coupled to the
solar collector and the bioreactor. In some embodiments, light from
the light source includes artificial light, wherein the artificial
light is produced by a light emitted diode (LED) or a fluorescent
light. In some embodiments, the system further includes a power
supply sufficient to power the LED or fluorescent light, wherein
the power supply is operatively coupled to the bioreactor; and a
light controller operatively coupled to the power supply, wherein
the light controller is adapted to control the intensity and
wavelength of light emitted by the LED or fluorescent light. In
some embodiments, the optical fibre is mounted in a transparent and
protective light structure. In some embodiments, the LED is mounted
in a transparent and protective light structure.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0022] These and other features, aspects, and advantages of the
present invention will become better understood with regard to the
following description, and accompanying drawings, where:
[0023] FIG. 1 is one embodiment of a bioreactor.
[0024] FIG. 2 illustrates the isoprenoid/carotenoid pathway.
[0025] FIGS. 3A-C show the growth of UTEX 1185 under white, blue,
and red light conditions. The X-axis is shown in days. Glu stands
for glucose.
[0026] FIGS. 4A-C show the growth of UTEX 2629 under white, blue,
and red light conditions. The X-axis is shown in days. Glu stands
for glucose.
[0027] FIGS. 5A-C show the growth of UTEX 2441 under white, blue,
and red light conditions. Glu stands for glucose.
[0028] FIG. 6 shows the production of lipid by UTEX 2441 under red
light conditions. LG stands for light+glucose; DG stands for
dark+glucose.
[0029] FIG. 7 shows the growth of UTEX 2243 under white light
conditions.
DETAILED DESCRIPTION
[0030] Terms used in the claims and specification are defined as
set forth below unless otherwise specified.
[0031] "Axenic" means a culture of an organism that is free from
contamination by other living organisms.
[0032] "Biodiesel" is a biologically produced fatty acid alkyl
ester suitable for use as a fuel in a diesel engine.
[0033] The term "biomass" refers to material produced by growth
and/or propagation of cells. Biomass may contain cells and/or
intracellular contents as well as extracellular material.
Extracellular material includes, but is not limited to, compounds
secreted by a cell.
[0034] "Bioreactor" means an enclosure or partial enclosure in
which cells are cultured, optionally in suspension. FIG. 1 is an
example of a bioreactor. "Photobioreactor" refers to a container,
at least part of which is at least partially transparent or
partially open, thereby allowing light to pass through, in which
one or more microalgae cells are cultured. Photobioreactors may be
closed, as in the instance of a polyethylene bag or Erlenmeyer
flask, or may be open to the environment, as in the instance of an
outdoor pond.
[0035] As used herein, a "catalyst" refers to an agent, such as a
molecule or macromolecular complex, capable of facilitating or
promoting a chemical reaction of a reactant to a product without
becoming a part of the product. A catalyst thus increases the rate
of a reaction, after which, the catalyst may act on another
reactant to form the product. A catalyst generally lowers the
overall activation energy required for the reaction such that it
proceeds more quickly or at a lower temperature. Thus reaction
equilibrium may be more quickly attained. Examples of catalysts
include enzymes, which are biological catalysts, heat, which is a
non-biological catalyst, and metal catalysts used in fossil oil
refining processes.
[0036] "Cellulosic material" means the products of digestion of
cellulose, including glucose and xylose, and optionally additional
compounds such as disaccharides, oligosaccharides, lignin,
furfurals and other compounds. Nonlimiting examples of sources of
cellulosic material include sugar cane bagasses, sugar beet pulp,
corn stover, wood chips, sawdust and switchgrass.
[0037] The term "co-culture", and variants thereof such as
"co-cultivate", refer to the presence of two or more types of cells
in the same bioreactor. The two or more types of cells may both be
microorganisms, such as microalgae, or may be a microalgal cell
cultured with a different cell type. The culture conditions may be
those that foster growth and/or propagation of the two or more cell
types or those that facilitate growth and/or proliferation of one,
or a subset, of the two or more cells while maintaining cellular
growth for the remainder.
[0038] The term "cofactor" is used herein to refer to any molecule,
other than the substrate, that is required for an enzyme to carry
out its enzymatic activity.
[0039] The term "cultivated", and variants thereof, refer to the
intentional fostering of growth (increases in cell size, cellular
contents, and/or cellular activity) and/or propagation (increases
in cell numbers via mitosis) of one or more cells by use of
intended culture conditions. The combination of both growth and
propagation may be termed proliferation. The one or more cells may
be those of a microorganism, such as microalgae. Examples of
intended conditions include the use of a defined medium (with known
characteristics such as pH, ionic strength, and carbon source),
specified temperature, oxygen tension, carbon dioxide levels, and
growth in a bioreactor. The term does not refer to the growth or
propagation of microorganisms in nature or otherwise without direct
human intervention, such as natural growth of an organism that
ultimately becomes fossilized to produce geological crude oil.
[0040] As used herein, the term "cytolysis" refers to the lysis of
cells in a hypotonic environment. Cytolysis is caused by excessive
osmosis, or movement of water, towards the inside of a cell
(hyperhydration). The cell cannot withstand the osmotic pressure of
the water inside, and so it explodes.
[0041] As used herein, the terms "expression vector" or "expression
construct" refer to a nucleic acid construct, generated
recombinantly or synthetically, with a series of specified nucleic
acid elements that permit transcription of a particular nucleic
acid in a host cell. The expression vector can be part of a
plasmid, virus, or nucleic acid fragment. Typically, the expression
vector includes a nucleic acid to be transcribed operably linked to
a promoter.
[0042] "Exogenous gene" refers to a nucleic acid transformed into a
cell. A transformed cell may be referred to as a recombinant cell,
into which additional exogenous gene(s) may be introduced. The
exogenous gene may be from a different species (and so
heterologous), or from the same species (and so homologous)
relative to the cell being transformed. In the case of a homologous
gene, it occupies a different location in the genome of the cell
relative to the endogenous copy of the gene. The exogenous gene may
be present in more than one copy in the cell. The exogenous gene
may be maintained in a cell as an insertion into the genome or as
an episomal molecule.
[0043] "Exogenously provided" describes a molecule provided to the
culture media of a cell culture.
[0044] "Fixed carbon source" means molecule(s) containing carbon,
e.g. organic, that are present at ambient temperature and pressure
in solid or liquid form.
[0045] "Homogenate" means biomass that has been physically
disrupted.
[0046] As used herein, "hydrocarbon" refers to: (a) a molecule
containing only hydrogen and carbon atoms wherein the carbon atoms
are covalently linked to form a linear, branched, cyclic, or
partially cyclic backbone to which the hydrogen atoms are attached;
or (b) a molecule that only primarily contains hydrogen and carbon
atoms and that can be converted to contain only hydrogen and carbon
atoms by one to four chemical reactions. Nonlimiting examples of
the latter include hydrocarbons containing an oxygen atom between
one carbon and one hydrogen atom to form an alcohol molecule, as
well as aldehydes containing a single oxygen atom. Methods for the
reduction of alcohols to hydrocarbons containing only carbon and
hydrogen atoms are well known. Another example of a hydrocarbon is
an ester, in which an organic group replaces a hydrogen atom (or
more than one) in an oxygen acid. The molecular structure of
hydrocarbon compounds varies from the simplest, in the form of
methane (CH.sub.4), which is a constituent of natural gas, to the
very heavy and very complex, such as some molecules such as
asphaltenes found in crude oil, petroleum, and bitumens.
Hydrocarbons may be in gaseous, liquid, or solid form, or any
combination of these forms, and may have one or more double or
triple bonds between adjacent carbon atoms in the backbone.
Accordingly, the term includes linear, branched, cyclic, or
partially cyclic alkanes, alkenes, lipids, and paraffin. Examples
include propane, butane, pentane, hexane, octane, triolein, and
squalene.
[0047] The term "hydrogen:carbon ratio" refers to the ratio of
hydrogen atoms to carbon atoms in a molecule on an atom-to-atom
basis. The ratio may be used to refer to the number of carbon and
hydrogen atoms in a hydrocarbon molecule. For example, the
hydrocarbon with the highest ratio is methane CH.sub.4 (4:1).
[0048] "Hydrophobic fraction" refers to the portion, or fraction,
of a material that is more soluble in a hydrophobic phase in
comparison to an aqueous phase. A hydrophobic fraction is
substantially insoluble in water and usually non-polar.
[0049] As used herein, the phrase "increase lipid yield" refers to
an increase in the productivity of a microbial culture by, for
example, increasing dry weight of cells per liter of culture,
increasing the percentage of cells that constitute lipid, or
increasing the overall amount of lipid per liter of culture volume
per unit time.
[0050] An "inducible promoter" is one that mediates transcription
of an operably linked gene in response to a particular
stimulus.
[0051] As used herein, the phrase "in operable linkage" refers to a
functional linkage between two sequences, such a control sequence
(typically a promoter) and the linked sequence. A promoter is in
operable linkage with an exogenous gene if it can mediate
transcription of the gene.
[0052] The term "in situ" means "in place" or "in its original
position". For example, a culture may contain a first microalga
secreting a catalyst and a second microorganism secreting a
substrate, wherein the first and second cell types produce the
components necessary for a particular chemical reaction to occur in
situ in the co-culture without requiring further separation or
processing of the materials.
[0053] A "limiting concentration of a nutrient" is a concentration
in a culture that limits the propagation of a cultured organism. A
"non-limiting concentration of a nutrient" is a concentration that
supports maximal propagation during a given culture period. Thus,
the number of cells produced during a given culture period is lower
in the presence of a limiting concentration of a nutrient than when
the nutrient is non-limiting. A nutrient is said to be "in excess"
in a culture, when the nutrient is present at a concentration
greater than that which supports maximal propagation.
[0054] As used herein, a "lipase" is a water-soluble enzyme that
catalyzes the hydrolysis of ester bonds in water-insoluble, lipid
substrates. Lipases catalyze the hydrolysis of lipids into
glycerols and fatty acids.
[0055] "Lipids" are a class of hydrocarbon that are soluble in
nonpolar solvents (such as ether and chloroform) and are relatively
or completely insoluble in water. Lipid molecules have these
properties because they consist largely of long hydrocarbon tails
which are hydrophobic in nature. Examples of lipids include fatty
acids (saturated and unsaturated); glycerides or glycerolipids
(such as monoglycerides, diglycerides, triglycerides or neutral
fats, and phosphoglycerides or 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).
"Fats" are a subgroup of lipids called "triacylglycerides."
[0056] The term "low irradiance of light" refers to the irradiance
of light that can be applied to a microorganism while avoiding
significant photoinhibition under heterotrophic conditions and the
irradiance of light needed to initiate a light-activated metabolism
in the microorganism. Light-activated metabolisms include, but are
not limited to, a life cycle, a circadian rhythm, cell division, a
biosynthetic pathway, and a transport system.
[0057] As used herein, the term "lysate" refers to a solution
containing the contents of lysed cells.
[0058] As used herein, the term "lysis" refers to the breakage of
the plasma membrane and optionally the cell wall of a biological
organism sufficient to release at least some intracellular content,
often by mechanical, viral or osmotic mechanisms that compromise
its integrity.
[0059] As used herein, the term "lysing" refers to disrupting the
cellular membrane and optionally the cell wall of a biological
organism or cell sufficient to release at least some intracellular
content.
[0060] "Microalgae" means a eukaryotic microbial organism that
contains a chloroplast, and optionally that is capable of
performing photosynthesis, or a prokaryotic microbial organism
capable of performing photosynthesis. Microalgae include obligate
photoautotrophs, which cannot metabolize a fixed carbon source as
energy, as well as heterotrophs, which can live solely off of a
fixed carbon source. Microalgae can refer to unicellular organisms
that separate from sister cells shortly after cell division, such
as Chlamydomonas, and can also refer to microbes such as, for
example, Volvox, which is a simple multicellular photosynthetic
microbe of two distinct cell types. "Microalgae" can also refer to
cells such as Chlorella and Dunaliella. "Microalgae" also includes
other microbial photosynthetic organisms that exhibit cell-cell
adhesion, such as Agmenellum, Anabaena, and Pyrobotrys.
"Microalgae" also includes obligate heterotrophic microorganisms
that have lost the ability to perform photosynthesis, such as
certain dinoflagellate algae species. Other examples of microalgae
are described below.
[0061] The terms "microorganism" and "microbe" are used
interchangeably herein to refer to microscopic unicellular
organisms, e.g., microalgae.
[0062] As used herein, the term "osmotic shock" refers to the
rupture of cells in a solution following a sudden reduction in
osmotic pressure. Osmotic shock is sometimes induced to release
cellular components of such cells into a solution.
[0063] "Polysaccharides" (also called "glycans") are carbohydrates
made up of monosaccharides joined together by glycosidic linkages.
Cellulose is an example of a polysaccharide that makes up certain
plant cell walls. Cellulose can be depolymerized by enzymes to
yield monosaccharides such as xylose and glucose, as well as larger
disaccharides and oligosaccharides.
[0064] "Port", in the context of a bioreactor, refers to an opening
in the bioreactor that allows influx or efflux of materials such as
gases, liquids, and cells. Ports are usually connected to tubing
leading from the bioreactor.
[0065] A "promoter" is defined as an array of nucleic acid control
sequences that direct transcription of a nucleic acid. As used
herein, a promoter includes necessary nucleic acid sequences near
the start site of transcription, such as, in the case of a
polymerase II type promoter, a TATA element. A promoter also
optionally includes distal enhancer or repressor elements, which
can be located as much as several thousand base pairs from the
start site of transcription.
[0066] As used herein, the term "recombinant" when used with
reference, e.g., to a cell, or nucleic acid, protein, or vector,
indicates that the cell, nucleic acid, protein or vector, has been
modified by the introduction of an exogenous nucleic acid or
protein or the alteration of a native nucleic acid or protein, or
that the cell is derived from a cell so modified. Thus, e.g.,
recombinant cells express genes that are not found within the
native (non-recombinant) form of the cell or express native genes
that are otherwise abnormally expressed, under expressed or not
expressed at all. By the term "recombinant nucleic acid" herein is
meant nucleic acid, originally formed in vitro, in general, by the
manipulation of nucleic acid, e.g., using polymerases and
endonucleases, in a form not normally found in nature. In this
manner, operably linkage of different sequences is achieved. Thus
an isolated nucleic acid, in a linear form, or an expression vector
formed in vitro by ligating DNA molecules that are not normally
joined, are both considered recombinant for the purposes of this
invention. It is understood that once a recombinant nucleic acid is
made and reintroduced into a host cell or organism, it will
replicate non-recombinantly, i.e., using the in vivo cellular
machinery of the host cell rather than in vitro manipulations;
however, such nucleic acids, once produced recombinantly, although
subsequently replicated non-recombinantly, are still considered
recombinant for the purposes of the invention. Similarly, a
"recombinant protein" is a protein made using recombinant
techniques, i.e., through the expression of a recombinant nucleic
acid as depicted above.
[0067] As used herein, the term. "renewable diesel" refers to
alkanes (such as C:10:0, C12:0, C:14:0, C16:0 and C18:0) produced
through hydrogenation and deoxygenation of lipids.
[0068] As used herein, the term "sonication" refers to a process of
disrupting biological materials, such as a cell, by use of sound
wave energy.
[0069] "Species of furfural" refers to 2-Furancarboxaldehyde or a
derivative thereof which retains the same basic structural
characteristics.
[0070] As used herein, "stover" refers to the dried stalks and
leaves of a crop remaining after a grain has been harvested.
[0071] "Wastewater" is watery waste which typically contains
washing water, laundry waste, feces, urine and other liquid or
semi-liquid wastes. It includes some forms of municipal waste as
well as secondarily treated sewage.
[0072] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
[0073] Microorganisms
[0074] Any species of organism that produces suitable lipid or
hydrocarbon can be used, although microorganisms that naturally
produce high levels of suitable lipid or hydrocarbon are preferred.
Production of hydrocarbons by microorganisms is reviewed by Metzger
et al. Appl Microbiol Biotechnol (2005) 66: 486-496 and A Look Back
at the U.S. Department of Energy's Aquatic Species Program:
Biodiesel from Algae, NREL/TP-580-24190, John Sheehan, Terri
Dunahay, John Benemann and Paul Roessler (1998).
[0075] Considerations affecting the selection of microorganisms for
use in the invention include, in addition to production of suitable
lipids or hydrocarbons for production of oils, fuels, and
oleochemicals, include: (1) high lipid content as a percentage of
cell weight; (2) ease of growth; (3) ease of genetic engineering;
and (4) ease of biomass processing. In particular embodiments, the
wild-type or genetically engineered microorganism yields cells that
are at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, or at least 70% or more lipid. Preferred
organisms grow heterotrophically or can be engineered to do so
using, for example, methods disclosed herein. The ease of
transformation and availability of selectable markers and
promoters, constitutive and/or inducible, that are functional in
the microorganism affect the ease of genetic engineering.
Processing considerations can include, for example, the
availability of effective means for lysing the cells.
[0076] In one embodiment, microorganisms include natural or
engineered microorganisms that can grow under heterotrophic
condition and use tight as signal to control cellular processes.
These can include alga such as Cyanophyta, Chlorophyta, Rhodophyta,
Cryptophyta, Chlorarachniophyta, Haptophyta, Euglenophyta,
Heterokontophyta, and Diatoms.
[0077] Algae
[0078] In one embodiment of the present invention, the
microorganism is a microalgae. Nonlimiting examples of microalgae
that can be used in accordance with the present invention are
described below.
[0079] More specifically, algal taxa belonging to the Cyanophyta,
including Cyanophyceae, are those being Prokaryotae, which have the
ability of oxygen evolution-type photosynthesis and are classified
into the following orders and families. Chroococcales include
Microcystaceae, Chroococcaceae, Entophysalidaceae,
Chamaesiphoniaceae, Dermocarpellaceae, Xenococcaceae, and
Hydrococcaceae, Oscillatoriales includes Borziaceae,
Pseudanabaenaceae, Schizotrichaceae, Phormidiaceae,
Oscillatoriaceae, and Homoeotrichaceae, Nostocales includes
Scytonemataceae, Microchaetaceae, Rivulariaceae, and Nostocaceae,
and Stigonematales includes Chlorogloeopsaceae, Capsosiraceae,
Stigonemataceae, Fischerellaceae Borzinemataceae, Nostochopsaceae,
and Mastigocladaceae.
[0080] Chlorophyta include Chlorophyceae, Prasinophyceae,
Pedinophyceae, Trebouxiophyceae, and Ulvophyceae. More
specifically, Chlorophyceae includes Acetabularia, Acicularia,
Actinochloris, Amphikrikos, Anadyomene, Ankistrodesmus, Ankyra,
Aphanochaete, Ascochloris, Asterococcus, Asteromonas, Astrephomene,
Atractomorpha, Axilococcus, Axilosphaera, Basichlamys, Basicladia,
Binuclearia, Bipedinomonas, Blastophysa, Boergesenia, Boodlea,
Borodinella, Borodinellopsis, Botryococcus, Brachiomonas,
Bracteacoccus, Bulbochaete, Caespitella, Capsosiphon, Carteria,
Centrosphaera, Chaetomorpha, Chaetonema, Chaetopeltis, Chaetophora,
Chalmasia, Chamaetrichon, Characiochloris, Characiosiphon,
Characium, Chlamydella, Chlamydobotrys, Chlamydocapsa,
Chlamydomonas, Chlamydopodium, Chloranomala, Chlorochydridion,
Chlorochytrium, Chlorocladus, Chlorocloster, Chlorococcopsis,
Chlorococcum, Chlorogonium, Chloromonas, Chlorophysalis,
Chlorosarcina, Chlorosarcinopsis, Chlorosphaera, Chlorosphaeropsis,
Chlorotetraedron, Chlorothecium, Chodatella, Choricystis,
Cladophora, Cladophoropsis, Cloniophora, Closteriopsis,
Coccobotrys, Coelastrella, Coelastropsis, Coelastrum, Coenochloris,
Coleochlamys, Coronastrum, Crucigenia, Crucigeniella, Ctenocladus,
Cylindrocapsa, Cylindrocapsopsis, Cylindrocystis, Cymopolia,
Cystococcus, Cystomonas, Dactylococcus, Dasycladus, Deasonia,
Derbesia, Desmatractum, Desmodesmus, Desmotetra, Diacanthos,
Dicellula, Dicloster, Dicranochaete, Dictyochloris, Dictyococcus,
Dictyosphaeria, Dictyosphaerium, Didymocystis, Didymogenes,
Dilabifilum, Dimorphococcus, Diplosphaera, Draparnaldia,
Dunaliella, Dysmorphococcus, Echinocoleum, Elakatothrix, Enallax,
Entocladia, Entransia, Eremosphaera, Ettlia, Eudorina,
Fasciculochloris, Fernandinella, Follicularia, Fottea, Franceia,
Friedmannia, Fritschiella, Fusola, Geminella, Gloeococcus,
Gloeocystis, Gloeodendron, Gloeomonas, Gloeotila, Golenkinia,
Gongrosira, Gonium, Graesiella, Granulocystis, Gyorffiana,
Haematococcus, Hazenia, Helicodictyon, Hemichloris,
Heterochlamydomonas, Heteromastix, Heterotetracystis,
Hormidiospora, Hormidium, Hormotila, Hormotilopsis, Hyalococcus,
Hyalodiscus, Hyalogonium, Hyaloraphidium, Hydrodictyon, Hypnomonas,
Ignatius, Interfilum, Kentrosphaera, Keratococcus, Kermatia,
Kirchneriella, Koliella, Lagerheimia, Lautosphaeria, Leptosiropsis,
Lobocystis, Lobomonas, Lola, Macrochloris, Marvania, Micractinium,
Microdictyon, Microspora, Monoraphidium, Muriella, Mychonastes,
Nanochlorum, Nautococcus, Neglectella, Neochloris, Neodesmus,
Neomeris, Neospongiococcum, Nephrochlamys, Nephrocytium,
Nephrodiella, Oedocladium, Oedogonium, Oocystella, Oocystis,
Oonephris, Ourococcus, Pachycladella, Palmella, Palmellococcus,
Palmellopsis, Palmodictyon, Pandorina, Paradoxia, Parietochloris,
Pascherina, Paulschulzia, Pectodictyon, Pediastrum, Pedinomonas,
Pedinopera, Percursaria, Phacotus, Phaeophila, Physocytium, Pilina,
Planctonema, Planktosphaeria, Platydorina, Platymonas, Pleodorina,
Pleurastrum, Pleurococcus, Ploeotila, Polyedriopsis, Polyphysa,
Polytoma, Polytomella, Prasinocladus, Prasiococcus, Protoderma,
Protosiphon, Pseudendocloniopsis, Pseudocharacium, Pseudochlorella,
Pseudochlorococcum, Pseudococcomyxa, Pseudodictyosphaerium,
Pseudodidymocystis, Pseudokirchneriella, Pseudopleurococcus,
Pseudoschizomeris, Pseudoschroederia, Pseudostichococcus,
Pseudotetracystis, Pseudotetradron, Pseudotrebouxia, Pteromonas,
Pulchrasphaera, Pyramimonas, Pyrobotrys, Quadrigula, Radiofilum,
Radiosphaera, Raphidocelis, Raphidonema, Raphidonemopsis,
Rhizoclonium, Rhopalosolen, Saprochaete, Scenedesmus,
Schizochlamys, Schizomeris, Schroederia, Schroederiella,
Scotiellopsis, Siderocystopsis, Siphonocladus, Sirogonium,
Sorastrum, Spermatozopsis, Sphaerella, Sphaerellocystis,
Sphaerellopsis, Sphaerocystis, Sphaeroplea, Spirotaenia,
Spongiochloris, Spongiococcum, Stephanoptera, Stephanosphaera,
Stigeoclonium, Struvea, Tetmemorus, Tetrabaena, Tetracystis,
Tetradesmus, Tetraedron, Tetrallantos, Tetraselmis, Tetraspora,
Tetrastrum, Treubaria, Triploceros, Trochiscia, Trochisciopsis,
Ulva, Uronema, Valonia, Valoniopsis, Ventricaria, Viridiella,
Vitreochlamys, Volvox, Volvulina, Westella, Willea, Wislouchiella,
Zoochlorella, Zygnemopsis, Hyalotheca, Chlorella,
Pseudopleurococcum and Rhopalocystis. Prasinophyceae includes
Heteromastix, Mammella, Mantoniella, Micromonas, Nephroselmis,
Ostreococcus, Prasinocladus, Prasinococcus, Pseudoscourfielda,
Pycnococcus, Pyramimonas, Scherffelia. Pedinophyceae includes
Marsupiomonas, Pedinomonas, Resultor. Trebouxiophyceae includes
Apatococcus, Asterochloris, Auxenochlorella, Chlorella, Coccomyxa,
Desmococcus, Dictyochloropsis, Elliptochloris, Jaagiella,
Leptosira, Lobococcus, Makinoella, Microthamnion, Myrmecia,
Nannochloris, Oocystis, Prasiola, Prasiolopsis, Prototheca,
Stichococcus, Tetrachlorella, Trebouxia, Trichophilus, Watanabea
and Myrmecia. Ulvophyceae includes Acrochaete, Bryopsis,
Cephaleuros, Chlorocystis, Enteromorpha, Gloeotilopsis,
Halochlorococcum, Ostreobium, Pirula, Pithophora, Planophila,
Pseudendoclonium, Trentepohlia, Trichosarcina, Ulothrix,
Bolbocoleon, Chaetosiphon, Eugomontia, Oltmannsiellopsis,
Pringsheimiella, Pseudodendroclonium, Pseudulvella, Sporocladopsis,
Urospora, and Wittrockiella.
[0081] Rhodophyta include Acrochaetium, Agardhiella, Antithamnion,
Antithamnionella, Asterocytis, Andouinella, Balbiania, Bangia,
Batrachospermum, Bonnemaisonia, Bostrychia, Callithamnion,
Caloglossa, Ceramium, Champia, Chroodactylon, Chroothece,
Compsopogon, Compsopogonopsis, Cumagloia, Cyanidium, Cystoclonium,
Dasya, Digenia, Dixoniella, Erythrocladia, Erythrolobas,
Erythrotrichia, Flintiella, Galdieria, Gelidium, Glaucosphaera,
Goniotrichum, Gracilaria, Grateloupia, Griffithsia, Hildenbrandia,
Hymenocladiopsis, Hypnea, Laingia, Membranoptera, Myriogramme,
Nemalion, Nemnalionopsis, Neoagardhiella, Palmaria, Phyllophora,
Polyneura, Polysiphonia, Porphyra, Porphyridium, Pseudochantransia,
Pterocladia, Pugetia, Rhodella, Rhodochaete, Rhodochorton,
Rhodosorus, Rhodospora, Rhodymenia, Seirospora, Selenastrum,
Sirodotia, Solieria, Spermothamnion, Spyridia, Stylonema, Thorea,
Trailiella and Tuomeya.
[0082] Cryptophyta include Cryptophyceae. More specifically,
Campylomonas, Chilomonas, Chroomonas, Cryptochrysis, Cryptomonas,
Goniomonas, Guillardia, Hanusia, Hemiselmis, Plagioselmis,
Proteomonas, Pyrenomonas, Rhodomonas and Stroreatula.
[0083] Chlorarachniophyta include Chlorarachnion, Lotharella and
Chattonella.
[0084] Haptophyta include Apistonema, Chrysochromulina,
Coccolithophora, Corcontochrysis, Cricosphaera, Diacronema,
Emiliana, Pavlova, Ruttnera, Cruciplacolithus, Prymnesium,
Isochrysis, Calyptrosphaera, Chrysotila, Coccolithus, Dicrateria,
Heterosigma, Hymenomonas, Imantonia, Gephyrocapsa, Ochrosphaera,
Phaeocystis, Platychrysis, Pseudoisochrysis, Syracosphaera and
Pleurochrysis.
[0085] Euglenophyta include stasia, Colacium, Cyclidiopsis,
Distigma, Euglena, Eutreptia, Eutreptiella, Gyropaigne,
Hyalophacus, Khawkinea Astasia, Lepocinclis, Menoidium, Parmidium,
Phacus, Rhabdomonas, Rhabdospira, Tetruetreptia and
Trachelomonas.
[0086] Heterokontophyta include Bacillariophyceae, Phaeophyceae,
Pelagophyceae, Xanthophyceae, Eustigmatophyceae, Syanurophyceae,
Phaeothamniophyceae and Raphidophyceae. More specifically,
Bacillariophyceae includes Achnanthes, Amphora, Chaetoceros,
Bacillaria, Nitzschia, Navicula, and Pinnularia. Phaeophyceae
includes Ascoseira, Asterocladon, Bodanella, Desmarestia,
Dictyocha, Dictyota, Ectocarpus, Halopteris, Heribaudiella,
Pleurocladia, Porterinema, Pylaiella, Sorocarpus, Spermatochnus,
Sphacelaria and Waerniella. Pelagophyceae includes Aureococcus,
Aureoumbra, Pelagococcus, Pelagomonas, Pulvinaria and
Sarcinochrysis. Xanthophyceae includes Chloramoebales,
Rhizochloridales, Mischococcales, Tribonematales, and Vaucheriales.
Eustigmatophyceae includes Chloridella, Ellipsoidion, Eustigmatos,
Monodopsis, Monodus, Nannochloropsis, Polyedriella,
Pseudocharaciopsis, Pseudostaurastrum and Vischeria Syanurophyceae
includes allomonas, Synura and Tessellaria. Phaeothamniophyceae
includes haeobotrys and Phacothamnion. Raphidophyceae includes
Olisthodiscus, Vacuolaria and Fibrocapsa.
[0087] Diatoms include Bolidophyceae, Coscinodiscophyceae,
Dinophyceae and Alveolates. Bolidophyceae include Bolidomonas,
Chrysophyceae, Giraudyopsis, Glossomastix, Chromophyton,
Chrysamoeba, Chrysochaete, Chrysodidymus, Chrysolepidomonas,
Chrysosaccus, Chrysosphaera, Chrysoxys, Cyclonexis, Dinobryon,
Epichrysis, Epipyxis, Hibberdia, Lagynion, Lepochromulina, Monas,
Monochrysis, Paraphysomonas, Phacoplaca, Phacoschizochlamys,
Picophagus, Pleurochrysis, Stichogloca and Uroglena.
Coscinodiscophyceae include Bacteriastrum, Bellerochea, Biddulphia,
Brockmanniella, Corethron, Coscinodiscus, Eucampia, Extubocellulus,
Guinardia, Helicotheca, Leptocylindrus, Leyanella, Lithodesmium,
Melosira, Minidiscus, Odentella, Planktoniella, Porosira,
Proboscia, Rhizosolenia, Stellarima, Thalassionema, Bicosoecid,
Symbiomonas, Actinocyclus, Amphora, Arcocellulus, Detonula,
Diatoma, Ditylum, Fragilariophyceae, Asterionellopsis, Delphineis,
Grammatophora, Nanofrustulum, Synedra and Tabularia. Dinophyceae
includes Adenoides, Alexandrium, Amphidinium, Ceratium,
Ceratocorys, Coolia, Crypthecodinium, Exuviaella, Gambierdiscus,
Gonyaulax, Gymnodinium, Gyrodinium, Heterocapsa, Katodinium,
Lingulodinium, Pfiesteria, Polarella, Protoceratium, Pyrocystis,
Scrippsiella, Symbiodinium, Thecadinium, Thoracosphaera, and
Zooxanthella. Alveolates include Cystodinium, Glenodinium,
Oxyrrhis, Peridinium, Prorocentrum, and Woloszynskia.
[0088] Methods of Culturing Microorganisms and Bioreactors
[0089] Microorganisms are generally cultured both for purposes of
conducting genetic manipulations and for subsequent production of
hydrocarbons (e.g., lipids, fatty acids, aldehydes, alcohols, and
alkanes). The former type of culture is generally conducted on a
small scale and initially, at least, under conditions in which the
starting microorganism can grow. Culture for purposes of
hydrocarbon production is usually conducted on a large scale.
Preferably a fixed carbon source (e.g. a feedstock) is present. The
culture can also be exposed to light some or all of the time.
[0090] Bioreactor
[0091] Microalgae can be cultured in liquid media. The culture can
be contained within a bioreactor. Microalgae can also be cultured
in photobioreactors that contain a fixed carbon source and allow
light to strike the cells. Exposure of microalgae cells to light,
even in the presence of a fixed carbon source that the cells
transport and utilize, can accelerate growth compared to culturing
cells in the dark. Culture condition parameters can be manipulated
to optimize total hydrocarbon production, the combination of
hydrocarbon species produced, and/or production of a hydrocarbon
species.
[0092] FIG. 1 is one embodiment of a bioreactor of the invention.
In one aspect a bioreactor is a photobioreactor. In one aspect, a
bioreactor system can be used for cultivating microalgae. The
bioreactor system can include a container and an irradiation
assembly, where the irradiation assembly is operatively coupled to
the container.
[0093] In one aspect, a bioreactor is a fermentation tank used for
industrial fermentation processes.
[0094] In some embodiments, a bioreactor includes glass, metal or
plastic tanks, equipped with, e.g., gauges and settings to control
aeration, stir rate, temperature, pH, and other parameters of
interest. Generally the gauges and settings are operatively coupled
to the bioreactor.
[0095] In one aspect, a bioreactor can be small enough for
bench-top applications (5-10 L or less) or up to 120,000 L or
larger in capacity for large-scale industrial applications.
[0096] In some embodiments, the bioreactor system can include a
light-diffusing structure or a plurality of light-diffusing
structures. In some embodiments, one or more of the light-diffusing
structures from the plurality of light-diffusing structures are
located along the interior surface of the bioreactor. In some
embodiments, the light-diffusing structure is operatively coupled
to the bioreactor.
[0097] The bioreactor system can include one or more optical fibers
and/or a plurality of light sources and/or a light source. In some
embodiments, the one or more optical fibres are mounted in
protective and optically transparent lighting structures. In some
embodiments, the optical fiber is operatively coupled to the
bioreactor. In some embodiments, the light source is operatively
coupled to the bioreactor.
[0098] In some embodiments, the bioreactor system can include a
lighting structure operatively coupled to a bioreactor. In certain
embodiments herein a lighting structure can have any shape or form
as it directs light signal to the interior of a bioreactor. The
bioreactor system may also include at least one optical fiber
extending from a first end of at least one of the one or more
optical fibers to a portion of a solar energy collector. In some
embodiments, the solar energy collector is operatively coupled to
the bioreactor. The optical fiber can be adapted to optically
couple the solar energy collector to the bioreactor. The optical
fiber may be optically coupled (directly or indirectly) to the
solar energy collector.
[0099] In some embodiments, the bioreactor system includes a
plurality of light sources operatively coupled to the bioreactor.
The plurality of light sources can include multiple LEDs. The
plurality of light sources comprising multiple LEDs can be operable
to supply full spectrum or a specific wavelength of artificial
light to a bioreactor.
[0100] In one embodiment, an LED is mounted in protective and
optically transparent lighting structures. In one embodiment the
LED is an array of LEDs.
[0101] In some embodiments, microalgae can be grown and maintained
in closed bioreactors made of different types of transparent or
semitransparent material. Such material can include Plexiglass.TM.
enclosures, glass enclosures, bags made from substances such as
polyethylene, transparent or semitransparent pipes, and other
materials. Microalgae can be grown and maintained in open
bioreactors such as raceway ponds, settling ponds, and other
non-enclosed containers.
[0102] The gas content of a bioreactor to grow microorganisms like
microalgae can be manipulated. Part of the volume of a bioreactor
can contain gas rather than liquid. Gas inlets can be used to pump
gases into the bioreactor. Any gas can be pumped into a bioreactor,
including air, air/O.sub.2 mixtures, noble gases such as argon and
others. The rate of entry of gas into a bioreactor can also be
manipulated. Increasing gas flow into a bioreactor increases the
turbidity of a culture of microalgae. Placement of ports conveying
gases into a bioreactor can also affect the turbidity of a culture
at a given gas flow rate. Air/O.sub.2 mixtures can be modulated to
generate optimal amounts of O.sub.2 for maximal growth by a
particular organism. Microalgae grow significantly faster in the
light under, for example, 3% O.sub.2/97% air than in 100% air. 3%
O.sub.2/97% air is approximately 100-fold more O.sub.2 than found
in air. For example, air:O.sub.2 mixtures of about 99.75% air:0.25%
O.sub.2, about 99.5% air:0.5% O.sub.2, about 99.0% air:1.00%
O.sub.2, about 98.0% air:2.0% O.sub.2, about 97.0% air:3.0%
O.sub.2, about 96.0% air:4.0% O.sub.2, and about 95.00% air:5.0%
O.sub.2 can be infused into a bioreactor or bioreactor.
[0103] Microalgae cultures can also be subjected to mixing using
devices such as spinning blades and impellers, rocking of a
culture, stir bars, infusion of pressurized gas, and other
instruments.
[0104] Bioreactors can have ports allowing entry of gases, solids,
semisolids and liquids into the chamber containing the microalgae.
Ports are usually attached to tubing or other means of conveying
substances. Gas ports, for example, convey gases into the culture.
Pumping gases into a bioreactor can serve to both feed cells
O.sub.2 and other gases and to aerate the culture and therefore
generate turbidity. The amount of turbidity of a culture varies as
the number and position of gas ports is altered. For example, gas
ports can be placed along the boftom of a cylindrical polyethylene
bag. Microalgae grow faster when O.sub.2 is added to air and
bubbled into a bioreactor.
[0105] Bioreactors preferably have one or more ports that allow
media entry. It is not necessary that only one substance enter or
leave a port. For example, a port can be used to flow culture media
into the bioreactor and then later can be used for sampling, gas
entry, gas exit, or other purposes. In some instances a bioreactor
is filled with culture media at the beginning of a culture and no
more growth media is infused after the culture is inoculated. In
other words, the microalgal biomass is cultured in an aqueous
medium for a period of time during which the microalgae reproduce
and increase in number; however quantities of aqueous culture
medium are not flowed through the bioreactor throughout the time
period. Thus in some embodiments, aqueous culture medium is not
flowed through the bioreactor after inoculation.
[0106] In other instances culture media can be flowed though the
bioreactor throughout the time period during which the microalgae
reproduce and increase in number. In some embodiments media is
infused into the bioreactor after inoculation but before the cells
reach a desired density. In other words, a turbulent flow regime of
gas entry and media entry is not maintained for reproduction of
microalgae until a desired increase in number of said microalgae
has been achieved.
[0107] Bioreactors preferably have one or more ports that allow gas
entry. Gas can serve to both provide nutrients such as O.sub.2 as
well as to provide turbulence in the culture media. Turbulence can
be achieved by placing a gas entry port below the level of the
aqueous culture media so that gas entering the bioreactor bubbles
to the surface of the culture. One or more gas exit ports allow gas
to escape, thereby preventing pressure buildup in the bioreactor.
Preferably a gas exit port leads to a "one-way" valve that prevents
contaminating microorganisms from entering the bioreactor. In some
instances cells are cultured in a bioreactor for a period of time
during which the microalgae reproduce and increase in number,
however a turbulent flow regime with turbulent eddies predominantly
throughout the culture media caused by gas entry is not maintained
for all of the period of time. In other instances a turbulent flow
regime with turbulent eddies predominantly throughout the culture
media caused by gas entry can be maintained for all of the period
of time during which the microalgae reproduce and increase in
number. In some instances a predetermined range of ratios between
the scale of the bioreactor and the scale of eddies is not
maintained for the period of time during which the microalgae
reproduce and increase in number. In other instances such a range
can be maintained.
[0108] Bioreactors preferably have at least one port that can be
used for sampling the culture. Preferably a sampling port can be
used repeatedly without altering compromising the axenic nature of
the culture. A sampling port can be configured with a valve or
other device that allows the flow of sample to be stopped and
started. Alternatively a sampling port can allow continuous
sampling. Bioreactors preferably have at least one port that allows
inoculation of a culture. Such a port can also be used for other
purposes such as media or gas entry.
[0109] In one embodiment, a bioreactor with an irradiation system
can be used to produce hydrocarbon from Botryococcus.
Botryococcenes are unbranched isoprenoid triterpenes having the
formula C.sub.nH.sub.2n-10. The A race produces alkadienes and
alkatrienes (derivatives of fatty acids) wherein n is an odd number
23 through 31. The B race produces botryococcenes wherein n is in
the range 30 through 40. These can be biofuels of choice for
hydrocracking to gasoline-type hydrocarbons.
[0110] Media
[0111] Microalgal culture media typically contains components such
as a fixed nitrogen source, trace elements, optionally a buffer for
pH maintenance, and phosphate. Other components can include a fixed
carbon source such as acetate or glucose, and salts such as sodium
chloride, particularly for seawater microalgae. Examples of trace
elements include zinc, boron, cobalt, copper, manganese, and
molybdenum in, for example, the respective forms of ZnCl.sub.2,
H.sub.3BO.sub.3, CoCl.sub.2.6H.sub.2O, CuCl.sub.2.2H.sub.2O,
MnCl.sub.2.4H.sub.2O and
(NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O.
[0112] For organisms able to grow on a fixed carbon source, the
fixed carbon source can be, for example, glucose, fructose,
sucrose, galactose, xylose, mannose, rhamnose, N-acetylglucosamine,
glycerol, floridoside, and/or glucuronic acid. The one or more
carbon source(s) can be supplied at a concentration of less than 50
.mu.M, at least about 50 .mu.M, at least about 100 .mu.M, at least
about 500 .mu.M, at least about 5 mM, at least about 50 mM, at
least about 500 mM, and more than 500 mM of one or more exogenously
provided fixed carbon source(s). The one or more carbon source(s)
can be supplied at a less than 1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,
22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,
35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,
48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,
61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100% of the media. The one or more carbon source(s) can also be
supplied at a percentage of the media between the above noted
percentages, e.g., 2.5% or 3.7% of the media.
[0113] Some microorganisms naturally grow on or can be engineered
to grow on a fixed carbon source that is a heterogeneous source of
compounds such as municipal waste, secondarily treated sewage,
wastewater, and other sources of fixed carbon and other nutrients
such as sulfates, phosphates, and nitrates. The sewage component
serves as a nutrient source in the production of hydrocarbons, and
the culture provides an inexpensive source of hydrocarbons.
[0114] Other culture parameters can also be manipulated, such as
the pH of the culture media, the identity and concentration of
trace elements and other media constituents.
[0115] Microorganisms useful in accordance with the methods of the
present invention are found in various locations and environments
throughout the world. As a consequence of their isolation from
other species and their resulting evolutionary divergence, the
particular growth medium for optimal growth and generation of lipid
and/or hydrocarbon constituents can vary. In some cases, certain
strains of microorganisms may be unable to grow on a particular
growth medium because of the presence of some inhibitory component
or the absence of some essential nutritional requirement required
by the particular strain of microorganism.
[0116] Solid and liquid growth media are generally available from a
wide variety of sources, and instructions for the preparation of
particular media that is suitable for a wide variety of strains of
microorganisms can be found, for example, online at a site
maintained by the University of Texas at Austin for its culture
collection of algae (UTEX).
[0117] Process conditions can be adjusted to increase the yield of
lipids suitable for a particular use and/or to reduce production
cost. For example, in certain embodiments, a microbe (e.g.,
microalgae) is cultured in the presence of a limiting concentration
of one or more nutrients, such as, for example, carbon and/or
nitrogen, phosphorous, or sulfur, while providing an excess of
fixed carbon energy such as glucose. Nitrogen limitation tends to
increase microbial lipid yield over microbial lipid yield in a
culture in which nitrogen is provided in excess. In particular
embodiments, the increase in lipid yield is at least about: 10%,
20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or 500%. The
microbe can be cultured in the presence of a limiting amount of a
nutrient for a portion of the total culture period or for the
entire period. In particular embodiments, the nutrient
concentration is cycled between a limiting concentration and a
non-limiting concentration at least twice during the total culture
period. [0118] Heterotrophic Growth and Light
[0119] Microorganisms can be cultured under heterotrophic growth
conditions in which a fixed carbon source provides energy for
growth and lipid accumulation.
[0120] Standard methods for the heterotrophic growth and
propagation of microalgae are known (see for example Miao and Wu, J
Biotechnology, 2004, 11:85-93 and Miao and Wu, Biosource Technology
(2006) 97:841-846).
[0121] For hydrocarbon production, cells, including recombinant
cells of the invention described herein, can be cultured or
fermented in large quantities. The culturing may be in large liquid
volumes, such as in suspension cultures as an example. Other
examples include starting with a small culture of cells which
expand into a large biomass in combination with cell growth and
propagation as well as hydrocarbon production. Bioreactors or steel
fermentors can be used to accommodate large culture volumes. A
bioreactor can include a fermentor. A fermentor similar those used
in the production of beer and/or wine can be suitable, as are
extremely large fermentors used in the production of ethanol.
[0122] Appropriate nutrient sources for culture in a fermentor are
provided. These include raw materials such as one or more of the
following: a fixed carbon source such as glucose, corn starch,
depolymerized cellulosic material, sucrose, sugar cane, sugar beet,
lactose, milk whey, or molasses; a fat source, such as fats or
vegetable oils; a nitrogen source, such as protein, soybean meal,
cornsteep liquor, ammonia (pure or in salt form), nitrate or
nitrate salt, or molecular nitrogen; and a phosphorus source, such
as phosphate salts. Additionally, a fermentor allows for the
control of culture conditions such as temperature, pH, oxygen
tension, and carbon dioxide levels. Optionally, gaseous components,
like oxygen or nitrogen, can be bubbled through a liquid culture.
Other Starch (glucose) sources such as wheat, potato, rice, and
sorghum. Other carbon sources include process streams such as
technical grade glycerol, black liquor, organic acids such as
acetate, and molasses. Carbon sources can also be provided as a
mixture, such as a mixture of sucrose and depolymerized sugar beet
pulp.
[0123] A fermentor can be used to allow cells to undergo the
various phases of their growth cycle. As an example, an inoculum of
hydrocarbon-producing cells can be introduced into a medium
followed by a lag period (lag phase) before the cells begin growth.
Following the lag period, the growth rate increases steadily and
enters the log, or exponential, phase. The exponential phase is in
turn followed by a slowing of growth due to decreases in nutrients
and/or increases in toxic substances. After this slowing, growth
stops, and the cells enter a stationary phase or steady state,
depending on the particular environment provided to the cells.
[0124] Hydrocarbon production by cells disclosed herein can occur
during the log phase or thereafter, including the stationary phase
wherein nutrients are supplied, or still available, to allow the
continuation of hydrocarbon production in the absence of cell
division.
[0125] Preferably, microorganisms grown using conditions described
herein and known in the art comprise at least about 20% by weight
of lipid, preferably at least about 40% by weight, more preferably
at least about 50% by weight, and most preferably at least about
60% by weight.
[0126] In an alternate heterotrophic growth method in accordance
with the present invention, microorganisms can be cultured using
depolymerized cellulosic biomass as a feedstock. Cellulosic biomass
(e.g., stover, such as corn stover) is inexpensive and readily
available; however, attempts to use this material as a feedstock
for yeast have failed. In particular, such feedstock has been found
to be inhibitory to yeast growth, and yeast cannot use the 5-carbon
sugars produced from cellulosic materials (e.g., xylose from
hemi-cellulose). By contrast, microalgae can grow on processed
cellulosic material. Accordingly, the invention provides a method
of culturing microalgae in the presence of a cellulosic material
and/or a 5-carbon sugar.
[0127] Suitable cellulosic materials include residues from
herbaceous and woody energy crops, as well as agricultural crops,
i.e., the plant parts, primarily stalks and leaves, not removed
from the fields with the primary food or fiber product. Examples
include agricultural wastes such as sugarcane bagasse, rice hulls,
corn fiber (including stalks, leaves, husks, and cobs), wheat
straw, rice straw, sugar beet pulp, citrus pulp, citrus peels;
forestry wastes such as hardwood and softwood thinnings, and
hardwood and softwood residues from timber operations; wood wastes
such as saw mill wastes (wood chips, sawdust) and pulp mill waste;
urban wastes such as paper fractions of municipal solid waste,
urban wood waste and urban green waste such as municipal grass
clippings; and wood construction waste. Additional cellulosics
include dedicated cellulosic crops such as switchgrass, hybrid
poplar wood, and miscanthus, fiber cane, and fiber sorghum.
Five-carbon sugars that are produced from such materials include
xylose.
[0128] In still another alternative heterotrophic growth method in
accordance with the present invention, which itself may optionally
be used in combination with the methods described above, sucrose,
produced by example from sugar cane or sugar beet, is used as a
feedstock.
[0129] Heterotrophic growth can include the use of both light and
fixed carbon source(s) for cells to grow and produce hydrocarbons.
Heterotrophic growth can be conducted in a photobioreactor.
[0130] Bioreactors can be exposed to one or more light sources to
provide microalgae with a light signal. A light signal can be
provided via light directed to a surface of the bioreactor by a
light source. Preferably the light source provides an intensity
that is sufficient for the cells to grow, but not so intense as to
cause oxidative damage or cause a photoinhibitive response. In some
instances a light source has a wavelength range that mimics or
approximately mimics the range of the sun. In other instances a
different wavelength range is used. Bioreactors can be placed
outdoors or in a greenhouse or other facility that allows sunlight
to strike the surface. In some embodiments, photon intensities for
species of the genus Botryococcus are between 25 and 500 .mu.mE
m.sup.-2 s.sup.-1 (see for example Photosynth Res. 2005 June;
84(1-3):21-7).
[0131] The number of photons striking a culture of microalgae cells
can be manipulated, as well as other parameters such as the
wavelength spectrum and ratio of dark:light hours per day.
Microalgae can also be cultured in natural light, as well as
simultaneous and/or alternating combinations of natural light and
artificial light. For example, microalgae can be cultured under
natural light during daylight hours and under artificial light
during night hours.
[0132] In one aspect of the invention, a microorganism is exposed
to about 0.1% to about 1% of light irradiance required for
photosynthesis, preferably about 0.3% to about 0.8% of light
irradiance required for photosynthesis by the organism. Typical
light irradiance can be between 0.1-300 .mu.mol photons m.sup.-2
s.sup.-1 including less than 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51 to 99, 100, 101 to 149, 150, 151, to 199, 200, 201 to 249,
250, or greater than 250 .mu.mol photons m.sup.-2 s.sup.-1. Light
irradiance can be about 0.01-1 .mu.mol photons m.sup.-2 s.sup.-1,
preferably between 1-10 .mu.mol photons m.sup.-2 s.sup.-1, or
between 10-100 .mu.mol photons m.sup.-2 s.sup.-1, or between
100-300 .mu.mol photons m.sup.-2 s.sup.-1, or between 100-300
.mu.mol photons m.sup.-2 s.sup.-1. Also included are light
irradiances between the above noted light irradiances, e.g., 1.1,
2.1, 2.5, or 3.5 .mu.mol photons m.sup.-2 s.sup.-1.
[0133] In one aspect, different light spectrums (e.g. 360-700 nm)
can be used. Light spectrums can be less than 300, 300, 350, 400,
450, 500, 550, 600, 650, 700, or 750 nm or more. Also included are
light spectrums between the above noted light spectrums, e.g., 360
or 440 nm.
[0134] In one embodiment, the irradiation can be applied in a
continuous manner. In another embodiment, the irradiation can be
applied in a cyclic pattern with an appropriate period of lighting
including but not limited to 12 h light:12 h dark or 16 h light:8 h
dark. Light patterns can include less than 1, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24
h of light and/or less than 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours (h) of
dark. Also included are light patterns between the above noted
light patterns, e.g., 7.5 h of light or 7.5 h of dark.
[0135] In one embodiment, the irradiation can be natural sunlight
collected by a solar collector and transmitted to the interior of a
bioreactor through an optical fiber.
[0136] In another embodiment, artificial light, such as light
emitted diodes (LEDs) or fluorescent light can be used as light
source. In another embodiment, natural sunlight and artificial
light can be used together.
[0137] In one embodiment, the irradiation is a full spectrum of
light.
[0138] In another embodiment, the irradiation is a specific
wavelength of light or a range of spectrum light transmitted
through a specific filter.
[0139] Methods of Recovering Lipids and Hydrocarbons
[0140] Hydrocarbons (e.g., lipids, fatty acids, aldehydes,
alcohols, and alkanes) produced by cells of the invention can be
harvested, or otherwise collected, by any convenient means. For
example, hydrocarbons secreted from cells can be centrifuged to
separate the hydrocarbons in a hydrophobic layer from contaminants
in an aqueous layer and optionally from any solid materials as a
precipitate in after centrifugation. Material containing cell or
cell fractions can be treated with proteases to degrade
contaminating proteins before or after centrifugation. In some
instances the contaminating proteins are associated, possibly
covalently, to hydrocarbons or hydrocarbon precursors which form
hydrocarbons upon removal of the protein. In other instances the
hydrocarbon molecules are in a preparation that also contains
proteins. Proteases can be added to hydrocarbon preparations
containing proteins to degrade proteins (for example, the protease
from Streptomyces griseus can be used (SigmaAldrich catalog number
P5147). After digestion, the hydrocarbons are preferably purified
from residual proteins, peptide fragments, and amino acids. This
purification can be accomplished, for example, by methods listed
above such as centrifugation and filtration.
[0141] Extracellular hydrocarbons can also be extracted in vivo
from living microalgae cells which are then returned to a
bioreactor by exposure of the cells, in an otherwise sterile
environment, to a non-toxic extraction solvent, followed by
separation of the living cells and the hydrophobic fraction of
extraction solvent and hydrocarbons, wherein the separated living
cells are then returned to a culture container such as a stainless
steel fermentor or photobioreactor (see Biotechnol Bioeng. 2004
Dec. 5; 88(5):593-600 and Biotechnol Bioeng. 2004 Mar. 5;
85(5):475-81).
[0142] Hydrocarbons can also be isolated by whole cell extraction.
The cells are first disrupted and then intracellular and cell
membrane/cell wall-associated hydrocarbons as well as extracellular
hydrocarbons can be collected from the whole cell mass, such as by
use of centrifugation as described above.
[0143] Various methods are available for separating hydrocarbons
and lipids from cellular lysates produced by the above methods. For
example, hydrocarbons can be extracted with a hydrophobic solvent
such as hexane (see Frenz et al. 1989, Enzyme Microb. Technol.,
11:717). Hydrocarbons can also be extracted using liquefaction (see
for example Sawayama et al. 1999, Biomass and Bioenergy 17:33-39
and Inoue et al. 1993, Biomass Bioenergy 6(4):269-274); oil
liquefaction (see for example Minowa et al. 1995, Fuel
74(12):1735-1738); and supercritical CO.sub.2 extraction (see for
example Mendes et al. 2003, Inorganica Chimica Acta
356:328-334).
[0144] Miao and Wu describe a protocol of the recovery of
microalgal lipid from a culture in which the cells were harvested
by centrifugation, washed with distilled water and dried by freeze
drying. The resulting cell powder was pulverized in a mortor and
then extracted with n-hexane. Miao and Wu, Biosource Technology
(2006) 97:841-846. [0145] Lysing Cells
[0146] Intracellular lipids and hydrocarbons produced in
microorganisms are, in some embodiments, extracted after lysing the
cells of the microorganism. Once extracted, the lipids and/or
hydrocarbons can be further refined to produce, e.g., oils, fuels,
or oleochemicals.
[0147] After completion of culturing, the microorganisms can be
separated from the fermentation broth. Optionally, the separation
is effected by centrifugation to generate a concentrated paste.
Centrifugation does not remove significant amounts of intracellular
water from the microorganisms and is not a drying step. The biomass
can then be washed with a washing solution (e.g., DI water) to get
rid of the fermentation broth and debris. Optionally, the washed
microbial biomass may also be dried (oven dried, lyophilized, etc.)
prior to cell disruption. Alternatively, cells can be lysed without
separation from some or all of the fermentation broth when the
fermentation is complete. For example, the cells can be at a ratio
of less than 1:1 v:v cells to extracellular liquid when the cells
are lysed.
[0148] Microorganisms containing a lipid and/or hydrocarbon can be
lysed to produce a lysate. As detailed herein, the step of lysing a
microorganism (also referred to as cell lysis) can be achieved by
any convenient means, including heat-induced lysis, adding a base,
adding an acid, using enzymes such as proteases and polysaccharide
degradation enzymes such as amylases, using ultrasound, mechanical
lysis, using osmotic shock, infection with a lytic virus, and/or
expression of one or more lytic genes. Lysis is performed to
release intracellular molecules which have been produced by the
microorganism. Each of these methods for lysing a microorganism can
be used as a single method or in combination simultaneously or
sequentially.
[0149] The extent of cell disruption can be observed by microscopic
analysis. Using one or more of the methods described herein,
typically more than 70% cell breakage is observed. Preferably, cell
breakage is more than 80%, more preferably more than 90% and most
preferred about 100%.
[0150] In particular embodiments, the microorganism is lysed after
growth, for example to increase the exposure of cellular lipid
and/or hydrocarbon for extraction or further processing. The timing
of lipase expression (e.g., via an inducible promoter) or cell
lysis can be adjusted to optimize the yield of lipids and/or
hydrocarbons. Below are described a number of lysis techniques.
These techniques can be used individually or in combination.
[0151] Heat-Induced Lysis
[0152] In a preferred embodiment of the present invention, the step
of lysing a microorganism comprises heating of a cellular
suspension containing the microorganism. In this embodiment, the
fermentation broth containing the microorganisms (or a suspension
of microorganisms isolated from the fermentation broth) is heated
until the microorganisms, i.e., the cell walls and membranes of
microorganisms degrade or breakdown. Typically, temperatures
applied are at least 50 C. Higher temperatures, such as, at least
30 C, at least 60 C, at least 70 C, at least 80 C, at least 90 C,
at least 100 C, at least 110 C, at least 120 C, at least 130 C or
higher are used for more efficient cell lysis.
[0153] Lysing cells by heat treatment can be performed by boiling
the microorganism. Alternatively, heat treatment (without boiling)
can be performed in an autoclave. The heat treated lysate may be
cooled for further treatment.
[0154] Cell disruption can also be performed by steam treatment,
i.e., through addition of pressurized steam. Steam treatment of
microalgae for cell disruption is described, for example, in U.S.
Pat. No. 6,750,048.
[0155] Lysis Using a Base
[0156] In another preferred embodiment of the present invention,
the step of lysing a microorganism comprises adding a base to a
cellular suspension containing the microorganism.
[0157] The base should be strong enough to hydrolyze at least a
portion of the proteinaceous compounds of the microorganisms used.
Bases which are useful for solubilizing proteins are known in the
art of chemistry. Exemplary bases which are useful in the methods
of the present invention include, but are not limited to,
hydroxides, carbonates and bicarbonates of lithium, sodium,
potassium, calcium, and mixtures thereof. A preferred base is KOH.
Base treatment of microalgae for cell disruption is described, for
example, in U.S. Pat. No. 6,750,048.
[0158] Acidic Lysis
[0159] In another preferred embodiment of the present invention,
the step of lysing a microorganism comprises adding an acid to a
cellular suspension containing the microorganism. Acid lysis can be
affected using an acid at a concentration of 10-500 mN or
preferably 40-160 nM. Acid lysis is preferably performed at above
room temperature (e.g., at 40-160, and preferably a temperature of
50-130. For moderate temperatures (e.g., room temperature to 100 C
and particularly room temperature to 65, acid treatment can
usefully be combined with sonication or other cell disruption
methods.
[0160] Lysing Cells Using Enzymes
[0161] In another preferred embodiment of the present invention,
the step of lysing a microorganism comprises lysing the
microorganism by using an enzyme. Preferred enzymes for lysing a
microorganism are proteases and polysaccharide-degrading enzymes
such as hemicellulase (e.g., hemicellulase from Aspergillus niger;
Sigma Aldrich, St. Louis, Mo.; #H2125), pectinase (e.g., pectinase
from Rhizopus sp.; Sigma Aldrich, St. Louis, Mo.; #P2401), Mannaway
4.0 L (Novozymes), cellulase (e.g., cellulose from Trichoderma
viride; Sigma Aldrich, St. Louis, Mo.; #C9422), and driselase
(e.g., driselase from Basidiomycetes sp.; Sigma Aldrich, St. Louis,
Mo.; #D9515.
[0162] Cellulases
[0163] In a preferred embodiment of the present invention, a
cellulase for lysing microorganism is a polysaccharide-degrading
enzyme, optionally from Chlorella or a Chlorella virus.
[0164] Proteases
[0165] Proteases such as Streptomyces griseus protease,
chymotrypsin, proteinase K, proteases listed in Degradation of
Polylactide by Commercial Proteases, Oda Y et al., Journal of
Polymers and the Environment, Volume 8, Number 1, January 2000, pp.
29-32(4), and other proteases can be used to lyse microorganisms.
Other proteases that can be used include Alcalase 2.4 FG
(Novozymes) and Flavourzyme 100 L (Novozymes).
[0166] Combinations
[0167] Any combination of a protease and a polysaccharide-degrading
enzyme can also be used, including any combination of the preceding
proteases and polysaccharide-degrading enzymes.
[0168] Lysing Cells Using Ultrasound
[0169] In another preferred embodiment of the present invention,
the step of lysing a microorganism is performed by using
ultrasound, i.e., sonication. Thus, cells can also by lysed with
high frequency sound. The sound can be produced electronically and
transported through a metallic tip to an appropriately concentrated
cellular suspension. This sonication (or ultrasonication) disrupts
cellular integrity based on the creation of cavities in cell
suspension.
[0170] Mechanical Lysis
[0171] In another preferred embodiment of the present invention,
the step of lysing a microorganism is performed by mechanical
lysis. Cells can be lysed mechanically and optionally homogenized
to facilitate hydrocarbon (e.g., lipid) collection. For example, a
pressure disrupter can be used to pump a cell containing slurry
through a restricted orifice valve. High pressure (up to 1500 bar)
is applied, followed by an instant expansion through an exiting
nozzle. Cell disruption is accomplished by three different
mechanisms: impingement on the valve, high liquid shear in the
orifice, and sudden pressure drop upon discharge, causing an
explosion of the cell. The method releases intracellular
molecules.
[0172] Alternatively, a ball mill can be used. In a ball mill,
cells are agitated in suspension with small abrasive particles,
such as beads. Cells break because of shear forces, grinding
between beads, and collisions with beads. The beads disrupt the
cells to release cellular contents. Cells can also be disrupted by
shear forces, such as with the use of blending (such as with a high
speed or Waring blender as examples), the french press, or even
centrifugation in case of weak cell walls, to disrupt cells.
[0173] Lysing Cells by Osmotic Shock (Cytolysis)
[0174] In another preferred embodiment of the present invention,
the step of lysing a microorganism is performed by applying an
osmotic shock.
[0175] Infection with a Lytic Virus
[0176] In a preferred embodiment of the present invention, the step
of lysing a microorganism comprises infection of the microorganism
with a lytic virus. A wide variety of viruses are known to lyse
microorganisms suitable for use in the present invention, and the
selection and use of a particular lytic virus for a particular
microorganism is within the level of skill in the art.
[0177] For example, paramecium bursaria chlorella virus (PBCV-1) is
the prototype of a group (family Phycodnaviridae, genus
Chlorovirus) of large, icosahedral, plaque-forming, double-stranded
DNA viruses that replicate in, and lyse, certain unicellular,
eukaryotic chlorella-like green algae. Accordingly, any susceptible
microalgae can be lysed by infecting the culture with a suitable
chlorella virus. See for example Adv. Virus Res. 2006; 66:293-336;
Virology, 1999 Apr. 25; 257(1):15-23; Virology, 2004 Jan. 5;
318(1):214-23; Nucleic Acids Symp. Ser. 2000; (44):161-2; J. Virol.
2006 March; 80(5):2437-44; and Annu. Rev. Microbiol. 1999;
53:447-94.
[0178] Autolysis (Expression of a Lytic Gene)
[0179] In another preferred embodiment of the present invention,
the step of lysing a microorganism comprises autolysis. In this
embodiment, a microorganism according to the invention is
genetically engineered to produce a lytic protein that will lyse
the microorganism. This lytic gene can be expressed using an
inducible promoter so that the cells can first be grown to a
desirable density in a fermentor, followed by induction of the
promoter to express the lytic gene to lyse the cells. In one
embodiment, the lytic gene encodes a polysaccharide-degrading
enzyme.
[0180] In certain other embodiments, the lytic gene is a gene from
a lytic virus. Thus, for example, a lytic gene from a Chlorella
virus can be expressed in an algal cell of the genus Chlorella,
such as C. protothecoides.
[0181] Suitable expression methods are described herein with
respect to the expression of a lipase gene. Expression of lytic
genes is preferably done using an inducible promoter, such as a
promoter active in microalgae that is induced by a stimulus such as
the presence of a small molecule, light, heat, and other stimuli.
For example, see Virology 260, 308-315 (1999); FEMS Microbiology
Letters 180 (1999) 45-53; Virology 263, 376-387 (1999); and
Virology 230, 361-368 (1997). [0182] Extraction of Lipids and
Hydrocarbons
[0183] Lipids and hydrocarbons generated by the microorganisms of
the present invention can be recovered by extraction with an
organic solvent. In some cases, the preferred organic solvent is
hexane. Typically, the organic solvent is added directly to the
lysate without prior separation of the lysate components. In one
embodiment, the lysate generated by one or more of the methods
described above is contacted with an organic solvent for a period
of time sufficient to allow the lipid anchor hydrocarbon components
to form a solution with the organic solvent. In some cases, the
solution can then be further refined to recover specific desired
lipid or hydrocarbon components. Hexane extraction methods are well
known in the art.
[0184] Methods of Processing Lipids and Hydrocarbons
[0185] Enzymatic Modification
[0186] Hydrocarbons (e.g., lipids, fatty acids, aldehydes,
alcohols, and alkanes) produced by cells as described herein can be
modified by the use of one or more enzymes, including a lipase.
When the hydrocarbons are in the extracellular environment of the
cells, the one or more enzymes can be added to that environment
under conditions in which the enzyme modifies the hydrocarbon or
completes its synthesis from a hydrocarbon precursor.
Alternatively, the hydrocarbons can be partially, or completely,
isolated from the cellular material before addition of one or more
catalysts such as enzymes. Such catalysts are exogenously added,
and their activity occurs outside the cell or in vitro.
[0187] Thermal and Other Catalytic Modification
[0188] Hydrocarbons produced by cells in vivo, or enzymatically
modified in vitro, as described herein can be optionally further
processed by conventional means. The processing can include
"cracking" to reduce the size, and thus increase the
hydrogen:carbon ratio, of hydrocarbon molecules. Catalytic and
thermal cracking methods are routinely used in hydrocarbon and
triglyceride oil processing. Catalytic methods involve the use of a
catalyst, such as a solid acid catalyst. The catalyst can be
silica-alumina or a zeolite, which result in the heterolytic, or
asymmetric, breakage of a carbon-carbon bond to result in a
carbocation and a hydride anion. These reactive intermediates then
undergo either rearrangement or hydride transfer with another
hydrocarbon. The reactions can thus regenerate the intermediates to
result in a self-propagating chain mechanism. Hydrocarbons can also
be processed to reduce, optionally to zero, the number of
carbon-carbon double, or triple, bonds therein. Hydrocarbons can
also be processed to remove or eliminate a ring or cyclic structure
therein. Hydrocarbons can also be processed to increase the
hydrogen:carbon ratio. This can include the addition of hydrogen
("hydrogenation") and/or the "cracking" of hydrocarbons into
smaller hydrocarbons.
[0189] Thermal methods involve the use of elevated temperature and
pressure to reduce hydrocarbon size. An elevated temperature of
about 800 C and pressure of about 700 kPa can be used. These
conditions generate "light," a term that is sometimes used to refer
to hydrogen-rich hydrocarbon molecules (as distinguished from
photon flux), while also generating, by condensation, heavier
hydrocarbon molecules which are relatively depleted of hydrogen.
The methodology provides homolytic, or symmetrical, breakage and
produces alkenes which may be optionally enzymatically saturated as
described above.
[0190] Catalytic and thermal methods are standard in plants for
hydrocarbon processing and oil refining. Thus hydrocarbons produced
by cells as described herein can be collected and processed or
refined via conventional means. See Hillen et al. (Biotechnology
and Bioengineering, Vol. XXIV: 193-205 (1982)) for a report on
hydrocracking of microalgae-produced hydrocarbons. In alternative
embodiments, the fraction is treated with another catalyst, such as
an organic compound, heat, and/or an inorganic compound. For
processing of lipids into biodiesel, a transesterification process
is used as described in Section IV herein.
[0191] Hydrocarbons produced via methods of the present invention
are useful in a variety of industrial applications. For example,
the production of linear alkylbenzene sulfonate (LAS), an anionic
surfactant used in nearly all types of detergents and cleaning
preparations, utilizes hydrocarbons generally comprising a chain of
10-14 carbon atoms. See, for example, U.S. Pat. Nos. 6,946,430;
5,506,201; 6,692,730; 6,268,517; 6,020,509; 6,140,302; 5,080,848;
and 5,567,359. Surfactants, such as LAS, can be used in the
manufacture of personal care compositions and detergents, such as
those described in U.S. Pat. Nos. 5,942,479; 6,086,903; 5,833,999;
6,468,955; and 6,407,044.
[0192] Methods of Producing Fuels Suitable for Use in Diesel
Vehicles and Jet Engines
[0193] Increasing interest is directed to the use of hydrocarbon
components of biological origin in fuels, such as biodiesel,
renewable diesel, and jet fuel, since renewable biological starting
materials that may replace stalling materials derived from fossil
fuels are available, and the use thereof is desirable. There is a
need for methods for producing hydrocarbon components from
biological materials. The present invention fulfills this need by
providing methods for production of biodiesel, renewable diesel,
and jet fuel using the lipids generated by the methods described
herein as a biological material to produce biodiesel, renewable
diesel, and jet fuel.
[0194] Traditional diesel fuels are petroleum distillates rich in
paraffinic hydrocarbons. They have boiling ranges as broad as 370
to 780 F, which are suitable for combustion in a compression
ignition engine, such as a diesel engine vehicle. The American
Society of Testing and Materials (ASTM) establishes the grade of
diesel according to the boiling range, along with allowable ranges
of other fuel properties, such as cetane number, cloud point, flash
point, viscosity, aniline point, sulfur content, water content, ash
content, copper strip corrosion, and carbon residue. Technically,
any hydrocarbon distillate material derived from biomass or
otherwise that meets the appropriate ASTM specification can be
defined as diesel fuel (ASTM D975), jet fuel (ASTM D1655), or as
biodiesel (ASTM D6751).
[0195] After extraction, lipid and/or hydrocarbon components
recovered from the microbial biomass described herein can be
subjected to chemical treatment to manufacture a fuel for use in
diesel vehicles and jet engines.
[0196] Biodiesel
[0197] Biodiesel is a liquid which varies in color--between golden
and dark brown--depending on the production feedstock. It is
practically immiscible with water, has a high boiling point and low
vapor pressure. Biodiesel refers to a diesel-equivalent processed
fuel for use in diesel-engine vehicles. Biodiesel is biodegradable
and non-toxic. An additional benefit of biodiesel over conventional
diesel fuel is lower engine wear.
[0198] Typically, biodiesel comprises C14-C18 alkyl esters. Various
processes convert biomass or a lipid produced and isolated as
described herein to diesel fuels. A preferred method to produce
biodiesel is by transesterification of a lipid as described herein.
A preferred alkyl ester for use as biodiesel is a methyl ester or
ethyl ester.
[0199] Biodiesel produced by a method described herein can be used
alone or blended with conventional diesel fuel at any concentration
in most modern diesel-engine vehicles. When blended with
conventional diesel fuel (petroleum diesel), biodiesel may be
present from about 0.1% to about 99.9%. Much of the world uses a
system known as the "B" factor to state the amount of biodiesel in
any fuel mix. For example, fuel containing 20% biodiesel is labeled
B20. Pure biodiesel is referred to as B100.
[0200] Biodiesel can also be used as a heating fuel in domestic and
commercial boilers. Existing oil boilers may contain rubber parts
and may require conversion to run on biodiesel. The conversion
process is usually relatively simple, involving the exchange of
rubber parts for synthetic parts due to biodiesel being a strong
solvent. Due to its strong solvent power, burning biodiesel will
increase the efficiency of boilers.
[0201] Biodiesel can be used as an additive in formulations of
diesel to increase the lubricity of pure Ultra-Low Sulfur Diesel
(ULSD) fuel, which is advantageous because it has virtually no
sulfur content.
[0202] Biodiesel is a better solvent than petrodiesel and can be
used to break down deposits of residues in the fuel lines of
vehicles that have previously been run on petrodiesel.
[0203] Production of Biodiesel
[0204] Biodiesel can be produced by transesterification of
triglycerides contained in oil-rich biomass. Thus, in another
aspect of the present invention a method for producing biodiesel is
provided. In a preferred embodiment, the method for producing
biodiesel comprises the steps of (a) cultivating a lipid-containing
microorganism using methods disclosed herein (b) lysing a
lipid-containing microorganism to produce a lysate, (c) isolating
lipid from the lysed microorganism, and (d) transesterifying the
lipid composition, whereby biodiesel is produced.
[0205] Methods for growth of a microorganism, lysing a
microorganism to produce a lysate, treating the lysate in a medium
comprising an organic solvent to form a heterogeneous mixture and
separating the treated lysate into a lipid composition have been
described above and can also be used in the method of producing
biodiesel.
[0206] Lipid compositions can be subjected to transesterification
to yield long-chain fatty acid esters useful as biodiesel.
Preferred transesterification reactions are outlined below and
include base catalyzed transesterification and transesterification
using recombinant lipases.
[0207] In a base-catalyzed transesterification process, the
triacylglycerides are reacted with an alcohol, such as methanol or
ethanol, in the presence of an alkaline catalyst, typically
potassium hydroxide. This reaction forms methyl or ethyl esters and
glycerin (glycerol) as a byproduct.
[0208] General Chemical Process
[0209] Animal and plant oils are typically made of triglycerides
which are esters of free fatty acids with the trihydric alcohol,
glycerol. In transesterification, the glycerol in a
triacylglyceride (TAG) is replaced with a short-chain alcohol such
as methanol or ethanol.
[0210] Using Recombinant Lipases
[0211] Transesterification has also been carried out experimentally
using an enzyme, such as a lipase instead of a base.
Lipase-catalyzed transesterification can be carried out, for
example, at a temperature between the room temperature and 80 C,
and a mole ratio of the TAG to the lower alcohol of greater than
1:1, preferably about 3:1.
[0212] Lipases suitable for use in transesterification are found
in, e.g. U.S. Pat. Nos. 4,798,793; 4,940,845 5,156,963; 5,342,768;
5,776,741 and WO89/01032.
[0213] One challenge to using a lipase for the production of fatty
acid esters suitable for biodiesel is that the price of lipase is
much higher than the price of sodium hydroxide (NaOH) used by the
strong base process. This challenge has been addressed by using an
immobilized lipase, which can be recycled. However, the activity of
the immobilized lipase must be maintained after being recycled for
a minimum number of cycles to allow a lipase-based process to
compete with the strong base process in terms of the production
cost. Immobilized lipases are subject to poisoning by the lower
alcohols typically used in transesterification. U.S. Pat. No.
6,398,707 (issued Jun. 4, 2002 to Wu et al.) describes methods for
enhancing the activity of immobilized lipases and regenerating
immobilized lipases having reduced activity.
[0214] In particular embodiments, a recombinant lipase is expressed
in the same microorganisms that produce the lipid on which the
lipase acts. DNA encoding the lipase and selectable marker is
preferably codon-optimized cDNA. Methods of recoding genes for
expression in microalgae are described in U.S. Pat. No.
7,135,290.
[0215] Standards
[0216] The common international standard for biodiesel is EN 14214.
ASTM D6751 is the most common biodiesel standard referenced in the
United States and Canada. Germany uses DIN EN 14214 and the UK
requires compliance with BS EN 14214.
[0217] Basic industrial tests to determine whether the products
conform to these standards typically include gas chromatography,
HPLC, and others. Biodiesel meeting the quality standards is very
non-toxic, with a toxicity rating (LD.sub.50) of greater than 50
mL/kg.
[0218] Renewable Diesel
[0219] Renewable diesel comprises alkanes, such as C16:0 and C18:0
and thus, are distinguishable from biodiesel. High quality
renewable diesel conforms to the ASTM D975 standard.
[0220] The lipids produced by the methods of the present invention
can serve as feedstock to produce renewable diesel. Thus, in
another aspect of the present invention, a method for producing
renewable diesel is provided. Renewable diesel can be produced by
at least three processes: hydrothermal processing (hydrotreating);
hydroprocessing; and indirect liquefaction. These processes yield
non-ester distillates. During these processes, triacylglycerides
produced and isolated as described herein, are converted to
alkanes.
[0221] In a preferred embodiment, the method for producing
renewable diesel comprises (a) cultivating a lipid-containing
microorganism using methods disclosed herein (b) lysing the
microorganism to produce a lysate, (c) isolating lipid from the
lysed microorganism, and (d) deoxygenating and hydrotreating the
lipid to produce an alkane, whereby renewable diesel is produced.
Lipids suitable for manufacturing renewable diesel can be obtained
via extraction from microbial biomass using an organic solvent such
as hexane, or via other methods, such as those described in U.S.
Pat. No. 5,928,696.
[0222] In some methods, the microbial lipid is first cracked in
conjunction with hydrotreating to reduce carbon chain length and
saturate double bonds, respectively. The material is then
isomerized, also in conjunction with hydrotreating. The naptha
fraction can then be removed through distillation, followed by
additional distillation to vaporize and distill components desired
in the diesel fuel to meet a D975 standard while leaving components
that are heavier than desired for meeting a D 975 standard.
Hydrotreating, hydrocracking, deoxygenation and isomerization
methods of chemically modifying oils, including triglyceride oils,
are well known in the art. See for example European patent
applications EP1741768 (A1); EP1741767 (A1); EP1682466 (A1);
EP1640437 (A1); EP1681337 (A1); EP1795576 (A1); and U.S. Pat. Nos.
7,238,277; 6,630,066; 6,596,155; 6,977,322; 7,041,866; 6,217,746;
5,885,440; 6,881,873.
[0223] Hydrotreating
[0224] In a preferred embodiment of the method for producing
renewable diesel, treating the lipid to produce an alkane is
performed by hydrotreating of the lipid composition. In
hydrothermal processing, typically, biomass is reacted in water at
an elevated temperature and pressure to form oils and residual
solids. Conversion temperatures are typically 300 to 660 F, with
pressure sufficient to keep the water primarily as a liquid, 100 to
170 standard atmosphere (atm). Reaction times are on the order of
15 to 30 minutes. After the reaction is completed, the organics are
separated from the water. Thereby a distillate suitable for diesel
is produced.
[0225] Hydroprocessing
[0226] A renewable diesel, referred to as "green diesel," can be
produced from fatty acids by traditional hydroprocessing
technology. The triglyceride-containing oils can be hydroprocessed
either as co-feed with petroleum or as a dedicated feed. The
product is a diesel fuel that conforms to the ASTM D975
specification. Thus, in another preferred embodiment of the method
for producing renewable diesel, treating the lipid composition to
produce an alkane is performed by hydroprocessing of the lipid
composition.
[0227] In some methods of making renewable diesel, the first step
of treating a triglyceride is hydroprocessing to saturate double
bonds, followed by deoxygenation at elevated temperature in the
presence of hydrogen and a catalyst. In some methods, hydrogenation
and deoxygenation occur in the same reaction. In other methods
deoxygenation occurs before hydrogenation. Isomerization is then
optionally performed, also in the presence of hydrogen and a
catalyst. Naphtha components are preferably removed through
distillation. For examples, see U.S. Pat. No. 5,475,160
(hydrogenation of triglycerides); U.S. Pat. No. 5,091,116
(deoxygenation, hydrogenation and gas removal); U.S. Pat. No.
6,391,815 (hydrogenation); and U.S. Pat. No. 5,888,947
(isomerization).
[0228] Petroleum refiners use hydroprocessing to remove impurities
by treating feeds with hydrogen. Hydroprocessing conversion
temperatures are typically 300 to 700 F. Pressures are typically 40
to 100 atm. The reaction times are typically on the order of 10 to
60 minutes.
[0229] Solid catalysts are employed to increase certain reaction
rates, improve selectivity for certain products, and optimize
hydrogen consumption.
[0230] Hydrotreating and hydroprocessing ultimately lead to a
reduction in the molecular weight of the feed. In the case of
triglyceride-containing oils, the triglyceride molecule is reduced
to four hydrocarbon molecules under hydroprocessing conditions: a
propane molecule and three heavier hydrocarbon molecules, typically
in the C8 to C18 range.
[0231] Indirect Liquefaction
[0232] A traditional ultra-low sulfur diesel can be produced from
any form of biomass by a two-step process. First, the biomass is
converted to a syngas, a gaseous mixture rich in hydrogen and
carbon monoxide. Then, the syngas is catalytically converted to
liquids. Typically, the production of liquids is accomplished using
Fischer-Tropsch (FT) synthesis. This technology applies to coal,
natural gas, and heavy oils. Thus, in yet another preferred
embodiment of the method for producing renewable diesel, treating
the lipid composition to produce an alkane is performed by indirect
liquefaction of the lipid composition.
[0233] Jet Fuel
[0234] Aeroplane fuel is clear to straw colored. The most common
fuel is an unleaded/paraffin oil-based fuel classified as Aeroplane
A-1, which is produced to an internationally standardized set of
specifications. Aeroplane fuel is a mixture of a large number of
different hydrocarbons, possibly as many as a thousand or more. The
range of their sizes (molecular weights or carbon numbers) is
restricted by the requirements for the product, for example,
freezing point or smoke point. Kerosone-type Aeroplane fuel
(including Jet A and Jet A-1) has a carbon number distribution
between about 8 and 16 carbon numbers. Wide-cut or naphta-type
Aeroplane fuel (including Jet B) typically has a carbon number
distribution between about 5 and 15 carbons.
[0235] Both Aeroplanes (Jet A and jet B) may contain a number of
additives. Useful additives include, but are not limited to,
antioxidants, antistatic agents, corrosion inhibitors, and fuel
system icing inhibitor (FSII) agents. Antioxidants prevent gumming
and usually, are based on alkylated phenols, for example, AO-30,
AO-31, or AO-37. Antistatic agents dissipate static electricity and
prevent sparking. Stadis 450 with dinonylnaphthylsulfonic acid
(DINNSA) as the active ingredient is an example. Corrosion
inhibitors, e.g., DCI-4A are used for civilian and military fuels
and DCI-6A is used for military fuels. FSII agents, include, e.g.,
Di-EGME.
[0236] A solution is blending algae fuels with existing jet fuel.
The present invention provides such a solution. The lipids produced
by the methods of the present invention can serve as feedstock to
produce jet fuel. Thus, in another aspect of the present invention,
a method for producing jet fuel is provided. Herewith two methods
for producing jet fuel from the lipids produced by the methods of
the present invention are provided: fluid catalytic cracking (FCC);
and hydrodeoxygenation (HDO).
[0237] Fluid Catalytic Cracking
[0238] Fluid Catalytic Cracking (FCC) is one method which is used
to produce olefins, especially propylene from heavy crude
fractions. There are reports in the literature that vegetable oils
such as canola oil could be processed using FCC to give a
hydrocarbon stream useful as a gasoline fuel.
[0239] The lipids produced by the method of the present invention
can be converted to olefins. The process involves flowing the
lipids produced through an FCC zone and collecting a product stream
comprised of olefins, which is useful as a jet fuel. The lipids
produced are contacted with a cracking catalyst at cracking
conditions to provide a product stream comprising olefins and
hydrocarbons useful as jet fuel.
[0240] In a preferred embodiment, the method for producing jet fuel
comprises (a) cultivating a lipid-containing microorganism using
methods disclosed herein, (b) lysing the lipid-containing
microorganism to produce a lysate, (c) isolating lipid from the
lysate, and (d) treating the lipid composition, whereby jet fuel is
produced.
[0241] In a preferred embodiment of the method for producing a jet
fuel, the lipid composition can be flowed through a fluid catalytic
cracking zone, which, in one embodiment, may comprise contacting
the lipid composition with a cracking catalyst at cracking
conditions to provide a product stream comprising C.sub.2-C.sub.5
olefins.
[0242] In certain embodiments of this method it may be desirable to
remove any contaminants that may be present in the lipid
composition. Thus, prior to flowing the lipid composition through a
fluid catalytic cracking zone, the lipid composition is pretreated.
Pretreatment may involve contacting the lipid composition with an
ion-exchange resin. The ion exchange resin is an acidic ion
exchange resin, such as Amberlyst.TM.-15 and can be used as a bed
in a reactor through which the lipid composition is flowed, either
upflow or downflow. Other pretreatments may include mild acid
washes by contacting the lipid composition with an acid, such as
sulfuric, acetic, nitric, or hydrochloric acid. Contacting is done
with a dilute acid solution usually at ambient temperature and
atmospheric pressure.
[0243] The lipid composition, optionally pretreated, is flowed to
an FCC zone where the hydrocarbonaceous components are cracked to
olefins. Catalytic cracking is accomplished by contacting the lipid
composition in a reaction zone with a catalyst composed of finely
divided particulate material. The reaction is catalytic cracking,
as opposed to hydrocracking, and is carried out in the absence of
added hydrogen or the consumption of hydrogen. As the cracking
reaction proceeds, substantial amounts of coke are deposited on the
catalyst. The catalyst is regenerated at high temperatures by
burning coke from the catalyst in a regeneration zone.
Coke-containing catalyst, referred to herein as "coked catalyst",
is continually transported from the reaction zone to the
regeneration zone to be regenerated and replaced by essentially
coke-free regenerated catalyst from the regeneration zone.
Fluidization of the catalyst particles by various gaseous streams
allows the transport of catalyst between the reaction zone and
regeneration zone. Methods for cracking hydrocarbons, such as those
of the lipid composition described herein, in a fluidized stream of
catalyst, transporting catalyst between reaction and regeneration
zones, and combusting coke in the regenerator are well known by
those skilled in the art of FCC processes. Exemplary FCC
applications and catalysts useful for cracking the lipid
composition to produce C.sub.2-C.sub.5 olefins are described in
U.S. Pat. Nos. 6,538,169, 7,288,685, which are incorporated in
their entirety by reference.
[0244] In one embodiment, cracking the lipid composition of the
present invention, takes place in the riser section or,
alternatively, the lift section, of the FCC zone. The lipid
composition is introduced into the riser by a nozzle resulting in
the rapid vaporization of the lipid composition. Before contacting
the catalyst, the lipid composition will ordinarily have a
temperature of about 149 C to about 316 C (300 F to 600 F). The
catalyst is flowed from a blending vessel to the riser where it
contacts the lipid composition for a time of abort 2 seconds or
less.
[0245] The blended catalyst and reacted lipid composition vapors
are then discharged from the top of the riser through an outlet and
separated into a cracked product vapor stream including olefins and
a collection of catalyst particles covered with substantial
quantities of coke and generally referred to as "coked catalyst."
In an effort to minimize the contact time of the lipid composition
and the catalyst which may promote further conversion of desired
products to undesirable other products, any arrangement of
separators such as a swirl arm arrangement can be used to remove
coked catalyst from the product stream quickly. The separator, e.g.
swirl arm separator, is located in an upper portion of a chamber
with a stripping zone situated in the lower portion of the chamber.
Catalyst separated by the swirl arm arrangement drops down into the
stripping zone. The cracked product vapor stream comprising cracked
hydrocarbons including light olefins and some catalyst exit the
chamber via a conduit which is in communication with cyclones. The
cyclones remove remaining catalyst particles from the product vapor
stream to reduce particle concentrations to very low levels. The
product vapor stream then exits the top of the separating vessel.
Catalyst separated by the cyclones is returned to the separating
vessel and then to the stripping zone. The stripping zone removes
adsorbed hydrocarbons from the surface of the catalyst by
counter-current contact with steam.
[0246] Low hydrocarbon partial pressure operates to favor the
production of light olefins. Accordingly, the riser pressure is set
at about 172 to 241 kPa (25 to 35 psia) with a hydrocarbon partial
pressure of about 35 to 172 kPa (5 to 25 psia), with a preferred
hydrocarbon partial pressure of about 69 to 138 kPa (10 to 20
psia). This relatively low partial pressure for hydrocarbon is
achieved by using steam as a diluent to the extent that the diluent
is 10 to 55 wt-% of lipid composition and preferably about 15 wt-%
of lipid composition. Other diluents such as dry gas can be used to
reach equivalent hydrocarbon partial pressures.
[0247] The temperature of the cracked stream at the riser outlet
will be about 510 C to 621 C (950 F to 1150 F). However, riser
outlet temperatures above 566 C (1050 F) make more dry gas and more
olefins. Whereas, riser outlet temperatures below 566 C (1050 F)
make less ethylene and propylene. Accordingly, it is preferred to
run the FCC process at a preferred temperature of about 566 C to
about 630 C, preferred pressure of about 138 kPa to about 240 kPa
(20 to 35 psia). Another condition for the process is the catalyst
to lipid composition ratio which can vary from about 5 to about 20
and preferably from about 10 to about 15.
[0248] In one embodiment of the method for producing a jet fuel,
the lipid composition is introduced into the lift section of an FCC
reactor. The temperature in the lift section will be very hot and
range from about 700 C (1292 F) to about 760 C (1400 F) with a
catalyst to lipid composition ratio of about 100 to about 150. It
is anticipated that introducing the lipid composition into the lift
section will produce considerable amounts of propylene and
ethylene.
[0249] Gas and liquid hydrocarbon products produced can be analyzed
by gas chromatography, HPLC, etc.
[0250] Hydrodeoxygenation
[0251] In another embodiment of the method for producing a jet fuel
using the lipid composition or the lipids produced as described
herein, the structure of the lipid composition or the lipids is
broken by a process referred to as hydrodeoxygenation (HDO).
[0252] HDO means removal of oxygen by means of hydrogen, that is,
oxygen is removed while breaking the structure of the material.
Olefinic double bonds are hydrogenated and any sulphur and nitrogen
compounds are removed. Sulphur removal is called
hydrodesulphurization (HDS). Pretreatment and purity of the raw
materials (lipid composition or the lipids) contribute to the
service life of the catalyst.
[0253] Generally in the HDO/HDS step, hydrogen is mixed with the
feed stock (lipid composition or the lipids) and then the mixture
is passed through a catalyst bed as a co-current flow, either as a
single phase or a two phase feed stock. After the HDO/MDS step, the
product fraction is separated and passed to a separate
isomerization reactor. An isomerization reactor for biological
starting material is described in the literature (FI 100 248) as a
co-current reactor.
[0254] The process for producing a fuel by hydrogenating a
hydrocarbon feed, e.g., the lipid composition or the lipids herein,
can also be performed by passing the lipid composition or the
lipids as a co-current flow with hydrogen gas through a first
hydrogenation zone, and thereafter the hydrocarbon effluent is
further hydrogenated in a second hydrogenation zone by passing
hydrogen gas to the second hydrogenation zone as a counter-current
flow relative to the hydrocarbon effluent. Exemplary HDO
applications and catalysts useful for cracking the lipid
composition to produce C.sub.2-C.sub.5 olefins are described in
U.S. Pat. No. 7,232,935, which is incorporated in its entirety by
reference.
[0255] Typically, in the hydrodeoxygenation step, the structure of
the biological component, such as the lipid composition or lipids
herein, is decomposed, oxygen, nitrogen, phosphorus and sulphur
compounds, and light hydrocarbons as gas are removed, and the
olefinic bonds are hydrogenated. In the second step of the process,
i.e. in the so-called isomerization step, isomerization is carried
out for branching the hydrocarbon chain and improving the
performance of the paraffin at low temperatures.
[0256] In the first step i.e. HDO step of the cracking process,
hydrogen gas and the lipid composition or lipids herein which are
to be hydrogenated are passed to a HDO catalyst bed system either
as co-current or counter-current flows, said catalyst bed system
comprising one or more catalyst bed(s), preferably 1-3 catalyst
beds. The HDO step is typically operated in a co-current manner. In
case of a HDO catalyst bed system comprising two or more catalyst
beds, one or more of the beds may be operated using the
counter-current flow principle.
[0257] In the HDO step, the pressure varies between 20 and 150 bar,
preferably between 50 and 100 bar, and the temperature varies
between 200 and 500 C, preferably in the range of 300-400 C.
[0258] In the HDO step, known hydrogenation catalysts containing
metals from Group VII and/or VIB of the Periodic System may be
used. Preferably, the hydrogenation catalysts are supported Pd, Pt,
Ni, NiMo or a CoMo catalysts, the support being alumina and/or
silica. Typically, NiMo/Al.sub.2O.sub.3 and CoMo/Al.sub.2O.sub.3
catalysts are used.
[0259] Prior to the HDO step, the lipid composition or lipids
herein may optionally be treated by prehydrogenation under milder
conditions thus avoiding side reactions of the double bonds. Such
prehydrogenation is carried out in the presence of a
prehydrogenation catalyst at temperatures of 50 400 C and at
hydrogen pressures of 1 200 bar, preferably at a temperature
between 150 and 250 C and at a hydrogen pressure between 10 and 100
bar. The catalyst may contain metals from Group VIII and/or VIB of
the Periodic System. Preferably, the prehydrogenation catalyst is a
supported Pd, Pt, Ni, NiMo or a CoMo catalyst, the support being
alumina and/or silica.
[0260] A gaseous stream from the HDO step containing hydrogen is
cooled and then carbon monoxide, carbon dioxide, nitrogen,
phosphorus and sulphur compounds, gaseous light hydrocarbons and
other impurities are removed therefrom. After compressing, the
purified hydrogen or recycled hydrogen is returned back to the
first catalyst bed and/or between the catalyst beds to make up for
the withdrawn gas stream. Water is removed from the condensed
liquid. The liquid is passed to the first catalyst bed or between
the catalyst beds.
[0261] After the HDO step, the product is subjected to an
isomerization step. It is substantial for the process that the
impurities are removed as completely as possible before the
hydrocarbons are contacted with the isomerization catalyst. The
isomerization step comprises an optional stripping step, wherein
the reaction product from the HDO step may be purified by stripping
with water vapour or a suitable gas such as light hydrocarbon,
nitrogen or hydrogen. The optional stripping step is carried out in
counter-current manner in a unit upstream of the isomerization
catalyst, wherein the gas and liquid are contacted with each other,
or before the actual isomerization reactor in a separate stripping
unit utilizing counter-current principle.
[0262] After the stripping step the hydrogen gas and the
hydrogenated lipid composition or lipids herein, and optionally an
n-paraffin mixture, are passed to a reactive isomerization unit
comprising one or several catalyst bed(s). The catalyst beds of the
isomerization step may operate either in co-current or
counter-current manner.
[0263] It is important for the process that the counter-current
flow principle is applied in the isomerization step. In the
isomerization step this is done by carrying out either the optional
stripping step or the isomerization reaction step or both in
counter-current manner.
[0264] The isomerization step and the HDO step may be carried out
in the same pressure vessel or in separate pressure vessels.
Optional prehydrogenation may be carried out in a separate pressure
vessel or in the same pressure vessel as the HDO and isomerization
steps.
[0265] In the isomerization step, the pressure varies in the range
of 20 150 bar, preferably in the range of 20 100 bar, the
temperature being between 200 and 500 C, preferably between 300 and
400 C.
[0266] In the isomerization step, isomerization catalysts known in
the art may be used. Suitable isomerization catalysts contain
molecular sieve and/or a metal from Group VII and/or a carrier.
Preferably, the isomerization catalyst contains SAPO-11 or SAPO41
or ZSM-22 or ZSM-23 or ferrierite and Pt, Pd or Ni and
Al.sub.2O.sub.3 or SiO.sub.2. Typical isomerization catalysts are,
for example, Pt/SAPO-11/Al.sub.2O.sub.3, Pt/ZSM-22/Al.sub.2O.sub.3,
Pt/ZSM-23/Al.sub.2O.sub.3 and Pt/SAPO-11/SiO.sub.2.
[0267] As the product, a high quality hydrocarbon component of
biological origin,useful as a diesel fuel or a component thereof,
is obtained, the density, cetane number and performance at low
temperate of said hydrocarbon component being excellent.
[0268] Microbe Engineering
[0269] As noted above, in certain embodiments of the present
invention it is desirable to genetically modify a microorganism to
enhance lipid production, modify the properties or proportions of
components generated by the microorganism, or to improve or provide
de novo growth characteristics on a variety of feedstock
materials.
[0270] Promoters, cDNAs, and 3'UTRs, as well as other elements of
the vectors, can be generated through cloning techniques using
fragments isolated from native sources (see for example Molecular
Cloning: A Laboratory Manual, Sambrook et al. (3d edition, 2001,
Cold Spring Harbor Press; and U.S. Pat. No. 4,683,202).
Alternatively, elements can be generated synthetically using known
methods (see for example Gene. 1995 Oct. 16; 164(1):49-53).
Microbial engineering methods are generally known in the art, e.g.,
U.S. Pat. App. No. 20090011480, herein incorporated by reference in
its entirety, for all purposes.
EXAMPLES
[0271] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way. Efforts have been made to ensure
accuracy with respect to numbers used (e.g., amounts, temperatures,
etc.), but some experimental error and deviation should, of course,
be allowed for.
[0272] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of protein chemistry,
biochemistry, recombinant DNA techniques and pharmacology, within
the skill of the art. Such techniques are explained fully in the
literature. See, e.g., T. E. Creighton, Proteins: Structures and
Molecular Properties (W.H. Freeman and Company, 1993); A. L.
Lehninger, Biochemistry (Worth Publishers, Inc., current addition);
Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd
Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan
eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences,
18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey
and Sundberg Advanced Organic Chemistry 3.sup.rd Ed. (Plenum Press)
Vols A and B (1992).
Example 1
Biomass Increase by Low Intensity-Illumination Application to
Microalgae Fermentation
[0273] Botryococcus naturally synthesizes and tolerates hydrocarbon
mixtures and produces as much as 85% hydrocarbon by weight, and in
many cases the major hydrocarbon is botryococcenes. It is also
known to be an obligate phototroph, but it seems to have the
ability to uptake glucose (Reference: "Biosynthesis of the
triterpenoids, botryococcenes and tetramethylsqualene in the B race
of Botryococcus braunii via the non-mevalonate pathway" Sato et al.
2003. Tetrahedron Letter 44:7035-7037).
[0274] Botryococcus culture is grown on BG11 media (Reference:
"Autotrophic cultivation of Botryococcus braunii for the production
of hydrocarbons and exopolysaccharides in various media". Dayananda
et al. 2007. Biomass and Bioenergy. 31: 87-93) at 25-35.degree. C.
in a bioreactor with 10-30% of dissolved oxygen. The effects of
light signal on heterotrophic growth of Botryococcus are tested by
comparing the cellular dry weight of the cultures from different
conditions (dark+no glucose, dark+glucose, light+no glucose, and
light+glucose). Optimum light intensity (0.01-300 .mu.mol photons
m.sup.-2 s.sup.-1) and different light spectrum (360-700 nm) as
well as different light periods (9-16 hr) are tested. Combination
of low irradiance of light and glucose results in a) improved
growth rate, b) increased products such as carotenoids, lipids, and
botryococcenes.
Example 2
Light Regulation of Isoprenoid Pathway
[0275] Isoprene, monoterpenes and sesquiterpenes are synthesized
and emitted by some plant and microalgal species, but not all
species have this ability. These volatile, nonessential isoprenoid
compounds share the same biochemical precursors as larger
commercially useful isoprenoids such as carotenoids and
hydrocarbons. Two separate pathways operate in plant cells to
synthesize prenyl diphosphate precursors common to all
isoprenoids.
[0276] Cytosolic and mitochondrial precursors are produced by the
mevalonic acid (MVA) pathway whereas the recently discovered
methylerythritol phosphate (MEP) pathway is located in plastids.
Botryococcus braunii produces hydrocarbon by the non-mevalonic, MEP
pathway (FIG. 2).
[0277] Light is the most important environmental factor for
regulation of MEP pathway. 1-deoxy-d-xylulose 5-phosphate
reductoisomerase (DXR) is the rate limiting step, and the
expression of the gene encoding DXR is regulated by light
(Reference: "Expression and molecular analysis of the Arabidopsis
DXR gene encoding 1-deoxy-d-xylulose 5-phosphate reductoisomerase,
the first committed enzyme of the 2-c-methyl-d-erythritol
4-phosphate pathway". Carretero-Paulet et al. Plant Physiology.
2002. 129:1581-1591).
[0278] Another example is blue-light activation of genes encoding
carotenoid biosynthetic enzymes in Chlamydomonas reinhardtii, an
unicellular green alga. Microarray and quantitative PCR experiments
showed the genes encoding carotenoid biosynthetic enzymes such as
PDS, HDS, PSY, and ZDS are activated by very low irradiance of
white light (0.01 .mu.mol photons m.sup.-2 s.sup.-1) and blue
light. Further evidence suggested phototropin, a blue light
receptor, is involved in blue-light activated gene expression for
carotenoid biosynthesis (Reference: "Phototropin involvement in the
expression of genes encoding chlorophyll and carotenoid
biosynthesis enzymes and LHC apoproteins in Chlamydomonas
reinhardtii". Im et al. The Plant Journal. 2006. 48:1-16).
[0279] One example is the increase of hydrocarbon production from
Botryococcus. Various light intensity (0.01-300 .mu.mol photons
m.sup.-2 s.sup.-1) and different light spectrum (360-700 nm) are
applied to a Botryococcus culture, and the amount of different
hydrocarbon species are measured by GC-MS.
Example 3
Growth of Neochloris oleabundans Under Different Light
Conditions
[0280] Materials and Methods
[0281] Microalgae and Culture Condition
[0282] Neochloris oleabundans strain UTEX 1185 was obtained from
the culture collection of algae at the University of Texas (Austin,
Tex. USA). Initial culture of the microalgae was grown in
Erlenmeyer 250 ml flask containing 120 ml modified bold 3 N medium
with 2% glucose at 25.degree. C. room temperature with an aluminum
foil loosely covering the flask on an orbital shaker at 130 rpm
under alternating two 40 W natural sunshine (392316, Philips) and
two 40 W plant and aquarium (392282, Philips) fluorescent light
bulbs. The culture medium (modified MB3N) contained the following
components per 1 L of deionized water: 0.75 g NaNO.sub.3, 0.075 g
K.sub.2HPO.sub.4, 0.074 g MgSO.sub.47H.sub.2O, 0.025 g
CaCl.sub.22H.sub.2O, 0.176 g KH.sub.2PO.sub.4, 0.025 g NaCl, 6 ml
of P-IV metal solution (0.75 g Na.sub.2EDTA2H.sub.2O, 0.097 g
FeCl.sub.36H.sub.2O, 0.041 g MnCl.sub.24H.sub.2O, 0.005 g
ZnCl.sub.2, 0.002 g CoCl.sub.26H.sub.2O, 0.004 g Na.sub.2MoO4
2H.sub.2O in 1 L dI water), 1 ml of each three vitamins (0.1 mM
vitamin B12, 0.1 mM biotin, 6.5 mM thiamine dissolved separately in
50 mM HEPES pH 7.8). Final pH of the medium was adjusted to 7.5
with 20% KOH before autoclaving the medium. The vitamin solutions
were added to cool down the autoclaved medium. Once the initial
culture reached certain confluence, its concentration was measured
using optical density (OD) at 680 nm and 750 nm using Genesys 10 UV
spectrophotometer (Thermo Scientific).
[0283] Experimental Procedure and Growth Measurement
[0284] Three different wavelengths of light (white, blue, and red)
were tested. LED lights were purchased from Super Bright LEDs, Inc.
(white: RL5-W3030, blue: RL5-B2430, red: RL5-R1330). For each light
wavelength, four different conditions were set up in duplicate as
follows:
[0285] 1-2. Modified MB3N+no glucose+dark
[0286] 3-4. Modified MB3N+no glucose+dim light
[0287] 5-6. Modified MB3N+2% glucose+dark
[0288] 7-8. Modified MB3N+2% glucose+dim light
[0289] A total of eight 250 ml Erlenmeyer flasks containing a final
volume of 120 ml cell culture were prepared with an initial cell
concentration of OD 0.1 at 750 nm (.about.1.1.times.10.sup.6
cells/ml) for each condition. Intensity of light was set at 3-4
.mu.mol/m.sup.2s.sup.-1 photons for white, 2-3
.mu.mol/m.sup.2s.sup.-1 photons for blue, and 1-2
.mu.mol/m.sup.2s.sup.-1 photons for red. The speed of the orbital
shaker was set at 135 rpm. The experiment was carried out at room
temperature for two weeks. 1 ml of cell culture was obtained from
each flask every 24 hrs to evaluate cell concentrations by
measuring OD at 680 nm and 750 nm using Genesys 10 UV
spectrophotometer from Thermo Fisher Scientific (Waltham, Mass.
USA). Specific growth rate was determined by plotting the logarithm
of culture optical density against time (FIG. 3). The combination
of a low irradiance of red, white, or blue light and glucose
resulted in an improved growth rate compared to controls.
Example 4
Growth of Botryococcus sudeticus Under Different Light
Conditions
[0290] Materials and Methods
[0291] Strains and Media
[0292] Botryococcus sudeticus strain UTEX 2629 was obtained from
the algae culture collection at the University of Texas (Austin,
Tex. USA). Stock culture was grown in Erlenmeyer 250 ml flasks
containing 120 ml modified BG11 medium with 2% glucose at
25.degree. C. room temperature with dim light (4-5
.mu.mol/m.sup.2s.sup.-1 photons) on an orbital shaker at 130 rpm.
Dim lighting is composed of two different bulbs, 40 W natural
sunshine (392316 Philips) and 40 W plant and aquarium fluorescent
light bulbs (392282 Philips). 1 L of culture medium (Modified
BG-11) contained: 10 mM HEPES (pH 7.8), 1.5 g NaNO.sub.3, 0.04 g
K.sub.2HPO.sub.4, 0.06 g MgSO.sub.47H.sub.2O, 0.036 g
CaCl.sub.22H.sub.2O, 0.006 g Citric acid H.sub.2O, 0.0138 g
Ammonium Ferric Citrate, 0.001 g Na.sub.2EDTA2H.sub.2O, 0.02 g
Na.sub.2CO.sub.3, 2.86 mg H.sub.3BO.sub.3, 1.81 mg
MnCl.sub.24H.sub.2O, 0.22 mg ZnSO.sub.47H.sub.2O, 0.39 mg
Na.sub.2MoO.sub.42H.sub.2O, 0.079 mg CuSO.sub.45H.sub.2O, 0.0494 mg
Co(NO.sub.3).sub.26H.sub.2O, 0.5 g casein hydrolysate, and 1 ml of
each three vitamins (0.1 mM vitamin B12, 0.1 mM biotin, 6.5 mM
thiamine dissolved separately in 50 mM HEPES pH 7.8). Final pH of
the medium was adjusted to 7.8 with 20% KOH.
[0293] Experimental Procedure and Growth Measurement
[0294] Three different wavelengths of light (white, blue, and red)
were tested. LED lights were purchased from Super Bright LEDs, Inc.
(white: RL5-W3030, blue: RL5-B2430, red: RL5-R1330). For each light
wavelength, four different conditions were set up in duplicate as
follows:
[0295] 1-2. Modified BG-11+no glucose+dark
[0296] 3-4. Modified BG-11+no glucose+dim light
[0297] 5-6. Modified BG-11+2% glucose+dark
[0298] 7-8. Modified BG-11+2% glucose+dim light
[0299] A total of eight 250 ml Erlenmeyer flasks containing a final
volume of 120 ml cell culture were prepared with an initial cell
concentration of OD 0.1 at 750 nm (.about.1.1.times.10.sup.6
cells/ml) for each condition. Intensity of light was set at 3-4
.mu.mol/m.sup.2s.sup.-1 photons for white, 2-3
.mu.mol/m.sup.2s.sup.-1 photons for blue, and 1-2
.mu.mol/m.sup.2s.sup.-1 photons for red. The speed of the orbital
shaker was set at 135 rpm. The experiment was carried out at room
temperature for two weeks. 1 ml of cell cultures was obtained from
each flask everyday to evaluate cell concentrations by measuring OD
at 680 nm and 750 nm using Genesys 10 UV spectrophotometer from
Thermo Fisher Scientific (Waltham, Mass. USA). Specific growth rate
was determined by plotting the logarithm of culture optical density
against time (FIG. 4). The combination of a low irradiance of red,
white, or blue light and glucose resulted in an improved growth
rate compared to controls.
Example 5
Botryococcus braunii: Fermentation with Controlled Illumination
[0300] Materials and Methods
[0301] Strains and Media
[0302] Botryococcus braunii strain UTEX 2441 was obtained from the
algae culture collection at the University of Texas (Austin, Tex.
USA). Stock culture was grown in Erlenmeyer 250 ml flasks
containing 120 ml modified BG11 medium with 2% glucose at
25.degree. C. room temperature with dim light (4-5
.mu.mol/m.sup.2s.sup.-1 photons) on an orbital shaker at 130 rpm.
Dim lighting was composed of two different bulbs, 40 W natural
sunshine (392316 Philips) and 40 W plant and aquarium fluorescent
light bulbs (392282 Philips). 1 L of culture medium (Modified
BG-11) contained: 10 mM HEPES (pH 7.8), 1.5 g NaNO.sub.3, 0.04 g
K.sub.2HPO.sub.4, 0.06 g MgSO.sub.47H.sub.2O, 0.036 g
CaCl.sub.22H.sub.2O, 0.006 g Citric acid H.sub.2O, 0.0138 g
Ammonium Ferric Citrate, 0.001 g Na.sub.2EDTA2H.sub.2O, 0.02 g
Na.sub.2CO.sub.3, 2.86 mg H.sub.3BO.sub.3, 1.81 mg
MnCl.sub.24H.sub.2O, 0.22 mg ZnSO.sub.47H.sub.2O, 0.39 mg
Na.sub.2MoO.sub.42H.sub.2O, 0.079 mg CuSO.sub.45H.sub.2O, 0.0494 mg
Co(NO.sub.3).sub.26H.sub.2O, 0.5 g casein hydrolysate, and 1 ml of
each three vitamins (0.1 mM vitamin B12, 0.1 mM biotin, 6.5 mM
thiamine dissolved separately in 50 mM HEPES pH 7.8). Final pH of
the medium was adjusted to 7.8 with 20% KOH.
[0303] Experimental Procedure and Growth Measurement
[0304] Three different wavelengths of lights (white, blue, and red)
were tested. LED lights were purchased from Super Bright LEDs, Inc.
(white: RL5-W3030, blue: RL5-B2430, red: RL5-R1330). For each light
wavelength, four different conditions were set up in duplicate as
follows:
[0305] 1-2 Modified BG-11+no glucose+dark
[0306] 3-4 Modified BG-11+no glucose+dim light
[0307] 5-6 Modified BG-11+2% glucose+dark
[0308] 7-8 Modified BG-11+2% glucose+dim light
[0309] A total of eight 250 ml Erlenmeyer flasks containing a final
volume of 120 ml cell culture were prepared with an initial cell
concentration of O.D 0.1 at 750 nm (.about.1.1.times.10.sup.6
cells/ml) for each condition. The intensity of light was set at 3-4
.mu.mol/m.sup.2s.sup.-3 photons for white, 2-3
.mu.mol/m.sup.2s.sup.-1 photons for blue, and 1-2
.mu.mol/m.sup.2s.sup.-1 photons far red. The speed of the orbital
shaker was set at 150 rpm. The experiment was carried out at room
temperature for two weeks. 5 ml of each cell culture was obtained
from each flask every two days to evaluate cell growth by dry cell
weight (DCW). Specific growth rate was determined by plotting the
culture DCW against time (FIG. 5).
[0310] Fluorescence Measurement of Neutral Lipid by Using Nile
Red
[0311] In 1 ml of algal suspension, 4 ul of Nile Red solution in
acetone (250 ug/ml) was added. The mixture was vortexed 2 times
during a 10 minute incubation at room temperature. After
incubation, 200 ul of stained algal samples were transferred into
individual wells in a 96-well plate. Fluorescence was measured on a
Molecular Devices 96 well plate spectrofluorometer with a 490 nm
excitation and 585 nm emission wavelength with 530 emission filter
cut off. In order to determine the relative fluorescence intensity
of algal samples, blank (Nile Red alone in the medium) was
subtracted from the fluorescence intensity.
[0312] Results
[0313] Red light+glucose increased the growth rate of UTEX 2441 by
35% compared to heterotrophic culture in the dark (Dark+glu) (FIG.
5). Lipid levels also increased by 52% under red light conditions
compared to controls (FIG. 6).
Example 6
Chlamydomonas reinhardtii: Fermentation with Controlled
Illumination
[0314] Materials and Methods
[0315] Strains and Media
[0316] Chlamydomonas reinhardtii strain UTEX 2243 was obtained from
the algae culture collection at the University of Texas (Austin,
Tex. USA). Stock cultures were grown separately in Erlenmeyer 250
ml flasks containing 120 ml TAP medium at 25.degree. C. room
temperature with dim light (4-5 .mu.mol/m.sup.2s.sup.-1 photons) on
an orbital shaker at 130 rpm. Dim lighting was composed of two
different bulbs (40 W natural sunshine (392316 Philips) and 40 W
plant and aquarium fluorescent light bulbs (392282 Philips)). 1 L
of culture medium (TAP) contained: 2.42 g Tris, 25 ml of TAP salts
solution (15 g NH4Cl, 4 g MgSO.sub.47H.sub.2O, 2 g
CaCl.sub.22H.sub.2O), 0.375 ml Phosphate solution (28.8 g
K.sub.2HPO.sub.4, 14.4 g KH2PO4 in 100 ml of water), 1 ml Hutner's
trace elements solution (1 L of Trace metal solution contains 50 g
EDTA disodium Salt, 22 g ZnSO.sub.47H.sub.2O, 11.4 g
H.sub.3BO.sub.4, 5.06 g MnCl.sub.24H.sub.2O, 1.61 g
CoCl.sub.26H.sub.2O, 1.57 g CuSO.sub.45H.sub.2O, 1.10
(NH.sub.4).sub.6Mo.sub.7O.sub.244H.sub.2O, 4.99 g
FeSO.sub.47H.sub.2O with pH 7.0 using either KOH or HCl) and 1 ml
of glacial acetic acid. Final pH of medium is 7.0 adjusted with
glacier acetic acid. Tris minimal medium (TP) was made with all
components listed above except acetic acid. The medium's pH was
adjusted to 7.0 with HCl.
[0317] Experimental Procedure and Growth Measurement
[0318] LED lights were purchased from Super Bright LEDs, Inc.
(RL5-W3030). Four different conditions were set up in duplicate as
follows:
[0319] 1-2. TP (no acetic acid)+dark
[0320] 3-4. TP (no acetic acid)+dim light
[0321] 5-6. TAP (acetic acid)+dark
[0322] 7-8. TAP (acetic acid)+dim light
[0323] A total of eight 250 ml Erlenmeyer flasks containing a final
volume of 120 ml cell culture were prepared with an initial cell
concentration of 1.0.times.10.sup.5 cells/ml for each condition.
The intensity of light was set at 3-5 .mu.mol/m.sup.2s.sup.-1
photons. The speed of the orbital shaker was set at 140 rpm. The
experiment was carried out at room temperature for one week. 500 ul
of cell culture was obtained from each flask every day to evaluate
cell growth by counting cell numbers. Cells were deflagellated
using lugol solution (1:20) prior to counting. Specific growth rate
was determined by plotting the cell number against time. The
combination of a low irradiance of white light and TAP resulted in
an improved growth rate compared to controls (FIG. 7).
Example 7
Cultivation of Microalgae with a Low Irradiance of Light
[0324] Materials and Methods
[0325] Strains and Media
[0326] Microalgae strains (e.g., Chlamydomonas, Botryococcus,
Neochloris, Cyanophyta, Chlorophyta, Rhodophyta, Cryptophyta,
Chlorarachniophyta, Haptophyta, Euglenophyta, Heterokontophyta,
Diatoms and/or those described in the description above) are
obtained from, e.g. the algae culture collection at the University
of Texas (Austin, Tex. USA). Stock culture is grown, e.g., in
Erlenmeyer 250 ml flasks containing the appropriate medium (see,
e.g., manufacturer's instructions) at about 25.degree. C. room
temperature with dim light (e.g., 4-5 .mu.mol/m.sup.2s.sup.-1
photons) on an orbital shaker at about 130 rpm. An appropriate
carbon source is used in the culture media, e.g., glucose. Dim
lighting can be composed of two different bulbs, e.g., 40 W natural
sunshine (392316 Philips) and 40 W plant and aquarium fluorescent
light bulbs (392282 Philips). The final pH of the medium is
adjusted as appropriate for the particular strain. See, e.g.,
manufacturer's instructions.
[0327] Experimental Procedure and Growth Measurement
[0328] Three different wavelengths of light (white, blue, and red)
are tested. LED lights are purchased from, e.g., Super Bright LEDs,
Inc. (white: RL5-W3030, blue: RL5-B2430, red: RL5-R1330). For each
light wavelength, four different conditions are set up in duplicate
as follows:
[0329] 1-2 no carbon+dark
[0330] 3-4 no carbon+dim light
[0331] 5-6 carbon+dark
[0332] 7-8 carbon+dim light
[0333] A total of eight 250 ml Erlenmeyer flasks containing a final
volume of 120 ml cell culture are prepared with an initial cell
concentration of, e.g., O.D 0.1 at 750 nm
(.about.1.1.times.10.sup.6 cells/ml) for each condition. Optimum
light intensity (e.g., 0.01-300 .mu.mol photons m.sup.-2 s.sup.-1)
and different light spectrums (e.g., 360-700 nm) as well as
different light periods (e.g., 9-16 hr of light) are tested. The
intensity of light is set at, e.g., 3-4 .mu.mol/m.sup.2s.sup.-1
photons for white, 2-3 .mu.mol/m.sup.2s.sup.-1 photons for blue,
and 1-2 .mu.mol/m.sup.2s.sup.-1 photons for red. Various carbon
sources at various concentrations are tested, e.g., glucose,
sucrose, fructose at a concentration of, e.g., 1%, 2%, or 3% of
culture media. The speed of the orbital shaker is set at, e.g., 150
rpm. The experiment is carried out at room temperature for less
than one, one, two, three, or more weeks. An aliquot of each cell
culture is obtained from each flask every one to two days to
evaluate cell growth by, e.g., dry cell weight (DCW). Specific
growth rate is determined by plotting the culture DCW against
time.
[0334] Measurement of Material of Interest
[0335] The amount of material of interest (hydrocarbon, lipid,
etc.) in the media is measured using standard means known in the
art, e.g., GC-MS or Nile Red as described above. For example, in 1
ml of algal suspension, 4 ul of Nile Red solution in acetone (250
ug/ml) is added. The mixture is vortexed during incubation at room
temperature. After incubation, 100-200 ul of stained algal samples
are transferred into individual wells in a 96-well plate.
Fluorescence is measured on, e.g., a Molecular Devices 96 well
plate spectrofluorometer with a 490 nm excitation and 585 nm
emission wavelength with 530 emission filter cut off. In order to
determine the relative fluorescence intensity of algal samples,
blank (Nile Red alone in the medium) is subtracted from the
fluorescence intensity.
[0336] Results
[0337] Red, white, and/or blue light in combination with a carbon
source increase the growth rate of the microalgae strain compared
to controls. Material of interest (e.g., hydrocarbon or lipid)
levels produced by the experimental microalgae strain (red, white,
and/or blue light in combination with a carbon source) increase
compared to controls.
[0338] While the invention has been particularly shown and
described with reference to a preferred embodiment and various
alternate embodiments, it will be understood by persons skilled in
the relevant art that various changes in form and details can be
made therein without departing from the spirit and scope of the
invention.
[0339] All references, issued patents and patent applications cited
within the body of the instant specification are hereby
incorporated by reference in their entirety, for all purposes.
* * * * *