U.S. patent application number 13/612650 was filed with the patent office on 2013-01-10 for continuous-batch hybrid process for production of oil and other useful products from photosynthetic microbes.
This patent application is currently assigned to HR Biopetroleum, Inc.. Invention is credited to Mark Edward Huntley, Donald G. Redalje.
Application Number | 20130011915 13/612650 |
Document ID | / |
Family ID | 37575789 |
Filed Date | 2013-01-10 |
United States Patent
Application |
20130011915 |
Kind Code |
A1 |
Huntley; Mark Edward ; et
al. |
January 10, 2013 |
Continuous-Batch Hybrid Process for Production of Oil and Other
Useful Products From Photosynthetic Microbes
Abstract
A process for cultivating photosynthetic microbes comprising
closed Systems for continuous cultivation and Open Systems for
batch cultivation, in which (a) the Closed System Area occupies no
more than 20% of the Total Land Area of the cultivation facility;
(b) batch cultures in the Open Systems are initiated with an
inoculum from the Closed Systems containing a cell biomass of no
less than 5% of the carrying capacity of said Open System; (c) the
doubling rate of said photosynthetic microbe is no less than once
every 16 hours; and (d) the residence time of the batch culture in
said Open System is no more than a period of 5 days.
Inventors: |
Huntley; Mark Edward;
(Kailua-Kona, HI) ; Redalje; Donald G.;
(Diamondhead, MS) |
Assignee: |
HR Biopetroleum, Inc.
Honolulu
HI
|
Family ID: |
37575789 |
Appl. No.: |
13/612650 |
Filed: |
September 12, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12806133 |
Aug 6, 2010 |
8268601 |
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13612650 |
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10582029 |
Jun 7, 2006 |
7770322 |
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PCT/US06/22443 |
Jun 7, 2006 |
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12806133 |
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60688396 |
Jun 7, 2005 |
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Current U.S.
Class: |
435/292.1 |
Current CPC
Class: |
C12M 23/18 20130101;
C12M 21/02 20130101; C12N 1/20 20130101; C12M 41/00 20130101; C12P
7/6463 20130101 |
Class at
Publication: |
435/292.1 |
International
Class: |
C12M 1/42 20060101
C12M001/42 |
Claims
1-13. (canceled)
14. A process for synthesizing oil, comprising: selecting
photosynthetic microbes that double in biomass in approximately 16
hours or less when supplied with sufficient carbon dioxide in an
open pond having a carrying capacity; injecting stack gases having
carbon dioxide concentrations of at least 3.5% into a
photobioreactor having a maximum culture depth of at most
approximately 30 to 40 centimeters, wherein said stack gases are
selected from the source consisting of burning a fossil fuel,
industrial production of chemicals, refining of oil and gas, and
geological extraction of fossil fuels; introducing said microbes
into said photobioreactor; providing said microbes with sufficient
nutrients in said photobioreactor to avoid nutrients being a
limiting factor to growth; turbulent mixing of said microbes
throughout said photobioreactor, whereby said microbes undergo
continuous exponential growth in said photobioreactor; inoculating
an initial biomass of said microbes that is no less than 5% of said
carrying capacity of said open pond from said closed
photobioreactor into said open pond; supplying carbon dioxide to
said open system to maintain approximately constant pH and to
supply sufficient carbon dioxide to said microbes and to replace
carbon dioxide removed by said microbes; and maintaining said
microbes in said open pond with high nutrient concentrations for at
most 5 days, whereby initial conditions of high light intensity and
high nutrient concentrations favor continued exponential growth for
a short period, but wherein growth becomes limited by nitrogen
availability which inhibits protein synthesis, whereby oil content
is increased.
15. A process for biosynthesis of oil, comprising: selecting
photosynthetic microbes that double in biomass in approximately 16
hours or less when supplied with sufficient carbon dioxide in an
open pond having a carrying capacity; dissolving stack gases having
carbon dioxide concentrations of at least 3.5% in a culture medium
in a photobioreactor having a maximum culture depth of at most
approximately 30 to 40 centimeters, wherein said stack gases are
selected from a source selected from the group consisting of
burning a fossil fuel, industrial production of chemicals, refining
of fossil fuels, and geological extraction of fossil fuels;
introducing said microbes into said medium in said photobioreactor;
providing said microbes with sufficient nutrients in said medium to
avoid nutrients being a limiting factor to growth; turbulent mixing
of said microbes in said medium throughout said photobioreactor to
provide a substantially homogeneous distribution of said microbes
in said medium, whereby said microbes undergo continuous
exponential growth in said photobioreactor; inoculating an initial
biomass of said microbes that is no less than 5% of said carrying
capacity of said open pond from said closed photobioreactor into
said open pond; supplying stack gases having carbon dioxide
concentrations of at least 3.5% to said open pond to maintain
approximately constant pH and to supply sufficient carbon dioxide
to said microbes and to replace carbon dioxide removed by said
microbes, wherein said stack gases are selected from a source
selected from the group consisting of burning a fossil fuel,
industrial production of chemicals, refining of fossil fuels, and
geological extraction of fossil fuels; and maintaining said
microbes in said open pond with high nutrient concentrations for at
most 5 days, whereby initial conditions of high light intensity and
high nutrient concentrations favor continued exponential growth for
a short period, but wherein growth becomes limited by nitrogen
availability which inhibits protein synthesis, whereby oil content
is increased.
16. A process for synthesizing biomass on a land area, comprising:
selecting photosynthetic microbes that double in biomass in
approximately 4 hours or less when supplied with sufficient carbon
dioxide in an open pond having a carrying capacity; introducing
said microbes into a photobioreactor having a maximum culture depth
of at most approximately 30 to 40 centimeters, wherein said
photobioreactor occupies less than 20% of said land area; providing
said microbes with sufficient nutrients in said photobioreactor to
avoid nutrients being a limiting factor to growth; turbulent mixing
of said microbes throughout said photobioreactor, whereby said
microbes undergo continuous exponential growth in said
photobioreactor; inoculating an initial biomass of said microbes
that is no less than 15% of said carrying capacity from said
photobioreactor into said open pond, wherein said open pond
occupies more than 80% of said land area; supplying carbon dioxide
to said open pond to maintain approximately constant pH and to
supply sufficient carbon dioxide to said microbes an to replace
carbon dioxide removed by said microbes; and maintaining said
microbes in said open pond with high nutrient concentrations for at
most 1 day, whereby initial conditions of high light intensity and
high nutrient concentrations favor continued exponential growth for
a short period, but wherein growth becomes limited by light due to
proliferation of cells.
17. A process for biosynthesis of oil, comprising: selecting
photosynthetic microbes that double in biomass in approximately 16
hours or less when supplied with sufficient carbon dioxide in an
open pond having a carrying capacity; dissolving stack gases having
carbon dioxide concentrations of at least 3.5% in a culture medium
in a photobioreactor having a maximum culture depth of at most
approximately 30 to 40 centimeters, wherein said stack gases are
selected from a source selected from the group consisting of
burning a fossil fuel, industrial production of chemicals, refining
of fossil fuels, and geological extraction of fossil fuels;
introducing said microbes into said medium in said photobioreactor;
providing said microbes with sufficient nutrients in said medium to
avoid nutrients being a limiting factor to growth; turbulent mixing
of said microbes in said medium throughout said photobioreactor,
whereby said microbes undergo continuous exponential growth in said
photobioreactor; inoculating an initial biomass of said microbes
that is no less than 5% of said carrying capacity of said open pond
from said closed photobioreactor into said open pond; supplying
carbon dioxide to said open pond to maintain approximately constant
pH and to supply sufficient carbon dioxide to said microbes and to
replace carbon dioxide removed by said microbes; and maintaining
said microbes in said open pond with high nutrient concentrations
for at most 5 days, whereby initial conditions of high light
intensity and high nutrient concentrations favor continued
exponential growth for a short period, but wherein growth becomes
limited by nitrogen availability which inhibits protein synthesis,
whereby oil content is increased.
18. A process for synthesizing biomass feedstock, comprising:
selecting photosynthetic microbes that double in biomass in
approximately 4 hours or less when supplied with sufficient carbon
dioxide in an open pond having a carrying capacity; dissolving
stack gases having carbon dioxide concentrations of at least 3.5%
in a culture medium in a photobioreactor having a maximum culture
depth of at most approximately 30 to 40 centimeters, wherein said
stack gases are selected from a source selected from the group
consisting of burning a fossil fuel, industrial production of
chemicals, refining of fossil fuels, and geological extraction of
fossil fuels; introducing said microbes into said medium in said
photobioreactor; providing said microbes with sufficient nutrients
in said medium to avoid nutrients being a limiting factor to
growth; turbulent mixing of said microbes in said medium throughout
said photobioreactor, whereby said microbes undergo continuous
exponential growth in said photobioreactor; inoculating an initial
biomass of said microbes that is no less than 15% of said carrying
capacity of said open pond from said closed photobioreactor into
said open pond; supplying carbon dioxide to said open pond to
maintain approximately constant pH and to supply sufficient carbon
dioxide to said microbes an to replace carbon dioxide removed by
said microbes; and maintaining said microbes in said open pond with
high nutrient concentrations for at most 1 day, whereby initial
conditions of high light intensity and high nutrient concentrations
favor continued exponential growth for a short period, but wherein
growth becomes limited by light due to proliferation of cells.
19. A device for cultivating photosynthetic microbes on a land
area, comprising: one or more culture vessels transparent to
visible light, each culture vessel providing a culture depth of at
most approximately 30 to 40 centimeters, wherein said culture
vessels cover a plan area of said land area and portions of said
land area between said culture vessels define an inert area; a
nutrient medium in each of said culture vessels; turbulent flow
means for creating turbulent flow throughout said nutrient medium
in each of said culture vessels, wherein said turbulent flow is
sufficiently low to prevent mechanical shear from damaging cells,
said culture vessels, said nutrient medium and said turbulent flow
means defining one or more photobioreactors; wherein said plan area
and said inert area occupy no more than 20% of said land area; and
one or more open ponds, each having a carrying capacity, which
together occupy no less than 80% of said land area; whereby a
biomass of microbes comprising at least 5% of said carrying
capacity for each of said open ponds can be inoculated from said
photobioreactors into said open ponds.
20. (canceled)
21. A process for synthesizing biomass on a land area, comprising:
selecting photosynthetic microbes that double in biomass in 16
hours or less when supplied with sufficient carbon dioxide in an
open pond having a carrying capacity; introducing said microbes
into a photobioreactor having a maximum culture depth of at most
approximately 30 to 40 centimeters, wherein said photobioreactor
occupies less than 20% of said land area; providing said microbes
with sufficient nutrients in said photobioreactor to avoid
nutrients being a limiting factor to growth; turbulent mixing of
said microbes throughout said photobioreactor, whereby said
microbes undergo continuous exponential growth in said
photobioreactor; inoculating an initial biomass of said microbes
that is no less than 5% of said carrying capacity from said
photobioreactor into said open pond, wherein said open pond
occupies more than 80% of said land area; supplying carbon dioxide
to said open pond to maintain approximately constant pH and to
replace carbon dioxide removed by said microbes; and maintaining
said microbes in said open pond with high nutrient concentrations
for at most 5 days, whereby initial conditions of high light
intensity and high nutrient concentrations favor continued
exponential growth for a short period, but wherein growth becomes
limited by light due to proliferation of cells.
22. A process according to claim 14, wherein said injecting step is
performed using a photobioreactor having a maximum culture depth of
at most approximately 15 to 20 centimeters.
23. A process according to claim 15, wherein said dissolving step
is performed using a photobioreactor having a maximum culture depth
of at most approximately 15 to 20 centimeters.
24. A process according to claim 16, wherein said introducing step
is performed using a photobioreactor having a maximum culture depth
of at most approximately 15 to 20 centimeters.
25. A process according to claim 17, wherein said dissolving step
is performed using a photobioreactor having a maximum culture depth
of at most approximately 15 to 20 centimeters.
26. A process according to claim 18, wherein said dissolving step
is performed using a photobioreactor having a maximum culture depth
of at most approximately 15 to 20 centimeters.
27. A process according to claim 19, wherein at least one of said
culture vessels has a culture depth of at most approximately 15 to
20 centimeters.
28. A process according to claim 21, wherein said introducing step
is performed using a photobioreactor having a maximum culture depth
of at most approximately 15 to 20 centimeters.
Description
TECHNICAL FIELD
[0001] This invention relates to a process for producing oils and
other useful products from photosynthetic microbes. The process
preferably utilizes large point sources of carbon dioxide caused by
industrial or resource extraction practices, such as stack gas from
fossil-fuel (coal, oil and gas) burning power plants, thus reducing
their emissions of carbon dioxide. The process produces, useful
products, including renewable fuel--plant oils that can be directly
made into liquid transportation fuel such as biodiesel.
[0002] The process is potentially very significant--for both
reducing global emissions of carbon dioxide, and for producing
biomass feedstocks as a starting material to manufacture fuels and
other useful products that are now made primarily from
geopetroleum. First, fossil-fuel burning power plants are now
responsible for about one-third of the world's emissions of carbon
dioxide--a so-called "greenhouse gas" that scientists believe is
primarily responsible for global warming. Major point sources of
carbon dioxide waste product include (i) stack gases from a variety
of industrial practices such as the production of chemicals and
refining of oil and gas, and (ii) collateral emissions created by
the geological extraction of oil and natural gas. Second, the
process described here employs microscopic plants to directly use
the very same carbon dioxide waste product created by burning fuels
to create yet more fuel, or to use the carbon dioxide waste product
created by chemical production to create yet more chemicals. Most
importantly, the use of microscopic plants in this process makes it
possible to use stack gases before they are emitted into the
atmosphere--a feat that cannot be accomplished by terrestrial
plant's--and thus reduces atmospheric emissions.
[0003] One of the main reasons that photosynthetic microbes are
superior to terrestrial plants as a source of feedstock for
biofuels and biologically-based chemical products is that they are
approximately 10 times more productive, per unit area. Low
productivity of terrestrial plants is a major bottleneck in the
production of biofuels, because feedstock material must be
transported to bioprocessing plants for such large distances that
the amount of fuel consumed by transportation alone can easily
exceed the amount of biofuel produced, thus limiting the
economically practical size of the bioprocessing plant. By using
photosynthetic microbes, which are about 10 times more productive,
bioprocessing plants can have about 10 times greater capacity
because the cost and fuel consumption of transporting the biomass
feedstock (the microbes) is reduced.
[0004] Power plant stack gases typically contain in the range of 5%
to 15% carbon dioxide, depending upon whether they are burning oil
or coal, respectively. Stack gas wastes from industrial production
processes contain comparably large amounts of carbon dioxide, and
waste streams generated during resource extraction may consist of
almost pure carbon dioxide. All plants use carbon dioxide and
Microscopic plants live in an aquatic medium, and can
withstand--indeed often require--carbon dioxide concentrations of
5% or more. Terrestrial plants, on the other hand, live in a
gaseous medium (the Earth's atmosphere), which currently has a
carbon dioxide concentration of about 0.035%, and cannot withstand
the hundred-fold higher concentrations found in stack gases.
[0005] Specifically, this invention relates to a two-stage
cultivation process, the "continuous-batch hybrid process," wherein
aqueous cultures of photosynthetic microbes (single-cell organisms,
including bacteria, cyanobacteria, and algae) are maintained in a
state of continuous exponential growth under nutrient-sufficient
conditions in a closed photobioreactor, from which a fraction is
periodically removed to inoculate a batch culture in an open
cultivation system where initial conditions of high light intensity
and high nutrient concentrations favor continued exponential growth
for a brief period, but wherein nutrients are rapidly exhausted and
light becomes the limiting factor due to the proliferation of
cells--conditions that favor oil biosynthesis, resulting in higher
cellular oil content. "Nutrients," as defined here, are comprised
of the so-called "Macronutrients," which are compounds of nitrogen
and phosphorus that are generally supplied in large quantities and
are the main food for growth, and the "Macronutrients," which
include vitamins and compounds containing trace metals like iron or
magnesium that are generally supplied in very small quantities.
Overall, the continuous-batch hybrid process yields higher oil
productivity than can be attained by a single stage process, either
continuous or batch. Additionally, the invention provides a
methodology for the reliable use of open cultivation systems, which
require far less skill and experience to operate than closed
systems, but which have proven otherwise unreliable for large-scale
cultivation.
[0006] Photosynthetic microbes are single-celled plants that, like
their multicellular (differentiated cells that are unable to
survive independently) terrestrial counterparts, produce biomass
that can be converted into fuels and other useful products that are
now derived almost exclusively from non-renewable fossil fuels.
Indeed, the fossilized ancestors of these photosynthetic microbes
are the dominant source of today's geological reserves of coal, oil
and gas. The biomass of today's photosynthetic microbes represent a
renewable feedstock for a variety of products that include, but are
not limited to oils, lubricants, plastics, petrochemicals, and
fuels. Photosynthetic microbes are potentially a much better source
of these products than terrestrial plants because they have higher
photosynthetic efficiency, grow about ten times faster, and thus
produce more biomass per unit area.
[0007] The oil content of photosynthetic microbes is generally
greatest under conditions that favor low growth rates. Conditions
that favor high growth rates and the highest oil content are
mutually exclusive. Any process that would yield both high growth
rates and high oil content in the shortest time would clearly yield
the highest overall rate of oil production. However, conditions
that favor high growth rates generally favor the highest content of
protein.
BACKGROUND ART
[0008] Thousands of species of photosynthetic microbes are
routinely cultivated at relatively small scale in the laboratory,
in culture vessels ranging from several milliliters up to a few
hundred liters in capacity. However, attempts to cultivate at
larger scales, generally necessary for commercial production, have
proven successful for fewer than 10 species--despite a worldwide
effort that has lasted half a century and consumed billions of
dollars.
[0009] There are two basic types of culture vessels that have been
employed in the effort to cultivate photosynthetic microbes at
commercial scale: (1) Photobioreactors (Closed Systems), and (2)
Open Systems.
[0010] (1) Closed Systems are characterized primarily by the
provision of means to control access to the atmosphere. Gas
exchange with the atmosphere is allowed to occur under controllable
conditions. Carbon dioxide enters the culture vessel as a fuel for
growth, and oxygen, the gaseous waste product of photosynthesis, is
permitted to escape the culture vessel. (The carbon is assimilated
into plant biomass, and the "dioxide"--oxygen--is expelled.)
However, gas exchange occurs through filtration mechanisms that are
designed to prohibit entry into the culture vessel of any species
of photosynthetic microbe other than the one that is being
preferentially cultivated therein.
[0011] Closed Systems are usually also designed to allow for the
control of other environmental conditions. The provision for
control of environmental variables such as temperature, pH,
nutrient concentrations, and light makes it possible to optimize
growth conditions for different species of microbial plants, which,
like terrestrial plants, have distinct preferences for unique
combinations of such variables.
[0012] For a given set of environmental conditions, all species of
photosynthetic microbes grow at their maximum rate within a narrow
range of cell concentrations. Accordingly, some Closed Systems are
designed to operate as "turbidostats," wherein the optical property
of turbidity (opaqueness), which is a function of cell
concentration, is monitored by means of programmable sensors that
measure the optical density of the medium. The operator may specify
a desired range of acceptable cell concentration, between a low
value and a high value. The low value corresponds directly to a
specific low optical density (the "low set point"), and the high
value to a specific high optical density (the "high set point").
The optical density sensor is then programmed accordingly. When
optical density attains a value that exceeds the designated upper
set point, the turbidostat activates a control mechanism that
provides for removing (harvesting) a fraction of the culture and
replacing it with cell-free nutrient medium, thereby diluting the
cell concentration to yield a value of optical density that matches
the designated lower set point. The cells then will grow,
increasing in concentration until optical density attains a value
that once again exceeds the upper set point, at which time the
cycle repeats.
[0013] Preferably a Closed System culture vessel is constructed
primarily of transparent material, such as glass or plastic, that
allows the transmission of photosynthetically active radiation
(visible light), but which otherwise separates the culture medium
from the atmosphere. Culture vessels may take many different
shapes, but they all share in common one spatial dimension that
limits their performance, and that is their depth relative to
incident light intensity. This feature arises from a basic property
of photosynthesis, namely, that photosynthetic rate is limited by
light intensity. Thus, at any given light intensity, the rate of
photosynthesis of a cell culture is maximized as a function of the
Lighted Area, i.e. the area of the culture medium that is exposed
to light (not necessarily the Surface Area, which may include areas
of the culture vessel that are not exposed to light). Consider two
culture vessels, both outdoors and exposed to sunlight. One is in
the shape of a rectangular pond with solid sides and bottom, and
the second is in the shape of a transparent cylinder, placed
horizontally on top of the ground. For the rectangular pond the
Surface Area includes the top, bottom, and sides of the pond, but
only the top area of the culture medium is exposed to sunlight. For
such an outdoor pond, then, the Lighted Area is equal to less than
half the Surface Area. By comparison, for the transparent cylinder,
the Surface Area is the entire surface of the cylinder and, no
matter what the time of day, half of the Surface Area will always
be directly illuminated by sunlight. Thus, for the cylinder, the
Lighted Area is equal to half the Surface Area
[0014] A second factor affecting the relationship between
photosynthesis and light is the cell concentration in the culture
medium. The greater the cell concentration within a medium, the
less the depth to which light may penetrate, because light
penetration decreases approximately exponentially as a function of
cell concentration. In other words, if the cell concentration
increases at a constant rate, light disappears faster and faster.
At some depth in a cell culture, then, light will actually decrease
to zero.
[0015] As a practical matter, the optimal culture depth for
photosynthetic microbes exposed to full sunlight is generally in
the range of 10 to 20 centimeters. No advantage can be gained by
providing greater depth of the culture, because the concentration
of cells per unit Lighted Area will remain the same, and deeper
cells will not receive enough light. The optimal depth, then, puts
a limit on the normal operating capacity of any culture system,
regardless of its Lighted Area. This phenomenon is a critically
important feature in the design of cultivation systems. Greater
volumes require more materials, at greater cost, but at some point
the increase in volume provides no increase in productivity per
unit Lighted Area.
[0016] Cultures of photosynthetic microbes generally require
stirring or mixing in order to maintain a homogeneous distribution
of cells in the medium. The natural tendency of photosynthetic
microbes in still water is to form dense aggregations, within which
the properties of the medium are altered to the detriment of the
culture. On a microscale, within the aggregation, the availability
of light and the concentration of nutrients and gases become so
different from the remainder of the medium that growth is limited.
Some species have appendages known as cilia or flagellae that allow
them to swim; such motile ("moving") species actively form
aggregations. Most non-motile species are heavier than water and
will sink, forming a passive aggregation on the bottom. To prevent
such aggregations, Closed Systems must provide a means for creating
turbulence using devices such as airlifts or pumps.
[0017] (2) Open Systems differ from Closed Systems in one critical
feature, namely that they are open to the atmosphere. This feature
is advantageous to both construction and operation, in several
ways. First, because the Lighted Area of an Open System is exposed
directly to sunlight, there is no requirement to use a transparent
material to construct the culture vessel; this affords broad
latitude in the choice of materials. Second, because no material is
used to cover the Lighted Area of the Open System, the amount and
cost of material is reduced by about half. Third, Open Systems are
generally easier to clean than Closed Systems. Over time the inner
surface of any culture vessel will tend to accumulate a film of
microbial growth. In a Closed System the accumulation of such a
film on the Lighted Area will absorb light; the consequent decrease
in light intensity causes a decrease in productivity. In both Open
Systems and Closed Systems the culture vessel surface can
accumulate microbial films of undesirable species that may be
detrimental to growth and production of the desired species. In
either case, the culture vessel surface will require cleaning from
time to time. As a practical matter. Open Systems allow a much
wider choice of cleaning methodologies. For example, people and
large types of mechanical cleaning equipment such as hoses,
pressure washers, and scrubbers that cannot enter the confined
space of a Closed System can easily enter an Open System.
[0018] The principal disadvantage of an Open System is that, by
being open to the atmosphere, it is susceptible to contamination by
unwanted species. One may begin the operation of an Open System
culture with only one desired species of photosynthetic microbe.
However, undesired species will inevitably be introduced, whether
by atmospheric transport or other means. Any undesired species that
grows faster than the desired species in the same environmental
conditions will, over time, outcompete the desired species and will
ultimately dominate the culture.
[0019] In summary, Closed Systems are designed specifically to
prohibit contamination by undesired species, with the expectation
that continuous cultivation of a desired species may be possible
for a much longer period than would be possible in an Open System.
However, Closed Systems are more complicated to construct and
operate. Open Systems afford a wider choice of materials for
construction, and also afford a wider choice of cleaning
methodologies. Closed Systems require additional operating
practices, such as the use of sterile technique during fluid
transfers, which call for greater time and expertise on the part of
the operator.
[0020] Theoretical differences between Closed Systems and Open
Systems have been borne out in practice. The first photosynthetic
microbe was isolated from nature and grown in pure culture little
more than a hundred years ago, but it was not until the late 1930s
that sufficiently large volumes of a single species could be
cultivated to permit chemical analysis. By the 1940s various
species were being grown in laboratory cultures of about 25 liters,
and it was discovered that, by altering environmental conditions of
the culture, either the oil or protein content of some species
could be made to exceed 60% of the total cell mass.
[0021] The first attempts at large-scale cultivation began in the
1950s, stimulated by widespread interest in photosynthetic microbes
as a source of cheap protein for foods and animal feeds. The first
Open Systems, built in Germany, took the shape of shallow,
elongated, recirculating raceways, with flow provided by a
paddlewheel device. Nationally-funded programs developed rapidly
throughout the world, all following the German "open pond" design.
The first open ponds had capacities of just a few thousand liters.
By the late 1950s, capacities of almost 100,000 liters had been
attained and, by the late 1960s, almost 1,000,000 liters. Such
increases in capacity brought economies of scale.
[0022] Hundreds of species were tested in the laboratory, and
attempts were made to grow the best protein producers in open ponds
during the 1960s and 1970s. Only a few species proved to be
amenable to sustained cultivation. These few species, such as
Spirulina platensis and Dunaliella salina, went on to become the
basis of commercial production, effected in open pond systems
covering hundreds of acres. The successful commercial species
proved to be "extremophiles," which thrive in conditions of
unusually high pH or salinity. Most species prefer conditions that
prevail in nature, where numerous species thrive simultaneously.
For two decades, all attempts to cultivate single-species cultures
of non-extremophiles in open ponds failed after less than a few
months because they were contaminated by other species that thrived
under the same environmental conditions.
[0023] Renewed interest in large-scale cultivation was stimulated
in the 1980s and 1990s by the prospect of producing renewable
biofuels using oils from photosynthetic microbes as a feedstock.
During this period government agencies of the USA and Japan, for
example, invested approximately $150 million in such an effort.
Such programs shared two goals: first, to collect and identify
species of photosynthetic microbes that produce high concentrations
of oil and then to determine the environmental conditions under
which they do so; and, second, to design and demonstrate the
operation of large-scale cultivation systems for the production of
biofuel feedstocks using species that had been developed in the
laboratory. Both programs succeeded at the first goal, but failed
at the second.
[0024] Laboratory studies quantified earlier findings. Culture
collections of hundreds of species were amassed. Research on
numerous strains demonstrated that, in general, nitrogen
sufficiency (nitrogen is needed for protein synthesis) promoted
high growth rates and low oil content, whereas nitrogen deficiency
resulted in low growth rates and high oil content. For some
species, it has also been noted that stress, caused by factors such
as high light intensity or very high temperatures, can induce
species to shift from protein synthesis to oil synthesis. Species
capable of optimal oil production--the highest oil content at the
highest growth rate--were selected for large-scale production
trials.
[0025] Large-scale production was once again attempted in the late
1980s and early 1990s using open pond systems. Operating results
were similar to those obtained for the three previous decades.
Promising oil-producing species were selected from the collections,
and cultures were inoculated into the ponds. However, as in prior
experience, single-species cultures could not be maintained for
more than a few weeks or months. The final report of the US program
referred to this phenomenon as an "uncertainty with the nature of
species control achieved."
[0026] By the 1990s the status of large-scale cultivation had not
progressed beyond the point reached in the 1960s. Three types of
microalgae--Spirulina, Dunaliella and Chlorella--were being
cultivated at facilities using open pond systems covering more than
100 acres. Scores of other species had been attempted worldwide,
but all attempts had failed. The biofuels programs, in particular,
had been unable to grow any desired species at any scale outside
the laboratory. Moreover, the biofuels programs had focused on
attempts to demonstrate the highest possible biomass production
rates under nutrient-sufficiency, conditions that are known from
laboratory studies to favor low oil content. No attempts were made
at large-scale to maximize oil production.
[0027] Large scale Closed System technology began to receive
significant attention in the early 1990s, once it became evident
that cultures of most species exposed to atmosphere were not
sustainable. At that time, the largest Closed Systems that had ever
been used were no more than a few thousand liters in capacity.
Advances in the past decade have succeeded at increasing reactor
capacity by a factor of about 10, to about 30,000 liters. But this
is nowhere near the rate of increase achieved for Open System
capacity that, also over a decade (in the 1950s to 1960s),
increased by a factor of 1,000.
[0028] The upper limit of Closed System capacity is, in large part,
a direct consequence of inherent design requirements. All basic
Closed System designs in use today were first developed in the
1950s, and may be categorized as follows: (1) vertical bags, tubes,
or towers; (2) flat-plate reactors; and (3) horizontal tubes.
Vertical systems are constrained by height limitations. Even when
exposed to full sunlight, most cultures achieve such high cell
densities that light is almost entirely absorbed at a distance of
more than 15 to 20 cm from the Lighted Area. This constraint limits
the diameter of the culture vessel to no more than 30 or 40 cm. To
achieve a capacity of more than 10,000 liters, for example, a 40-cm
diameter vertical system would have to be more than 80 meters (260
feet) high. Such dimensions present clear challenges in structural
engineering which, even if achievable, become increasingly complex
the greater the volume of the system. One of the obvious solutions
has been to introduce an illumination system within the reactor,
but experience has shown that this introduces other problems, of
which bio-fouling may be the greatest. Over a relatively short
time, the surface of the light source tends to become covered with
a microbial film, sharply reducing light intensity and thus
defeating the purpose of the light source. Removing the culture and
cleaning the vessel is one option, but hardly desirable if the goal
is sustained operation. Another common anti-fouling option, making
the surface of the light source toxic to microbes, is clearly
undesirable. In general, the use of internal illumination makes the
system more complex.
[0029] Horizontal systems such as flat-plate reactors and
horizontal tubes eliminate the need for the structural engineering
required of vertical systems. Using the earth's surface for
structural support, the potential capacity of such systems might
appear limitless. However, the capacity of horizontal systems is
generally limited by the requirement for turbulent flow, whether
used to maintain adequate mixing or to fill and empty the culture
vessel.
[0030] Turbulent flow in a pipe or a channel is described by the
Reynolds number, defined as the velocity of the fluid multiplied by
the "characteristic length" of the pipe or channel, and divided by
the viscosity of the fluid. The Reynolds number does not have any
units, like inches or pounds, and is therefore "dimensionless,"
like "one-half" or "two-thirds". The characteristic length of a
fluid-filled pipe is its diameter; the characteristic length of a
wide channel is its depth. For a fluid of constant viscosity, flow
will become increasingly turbulent as the velocity of flow
increases. Turbulence also increases in proportion to the
characteristic length; this happens because pipe and channel
surfaces are "sticky." Surfaces cause friction that slows down the
flow; the flow rate is almost zero next to the surface, and
increases with distance away from the surface. Thus, in a pipe or
channel with small characteristic length, the surface friction will
have a great effect on the average flow. By contrast, in a pipe or
channel with large characteristic length, the surface friction will
have little effect on the average flow, and turbulence will be
greater.
[0031] Surface friction also adds up over distance. Imagine a very
long pipe through which water is propelled by a pump. At the
origin, near the pump, the flow is turbulent. The farther the fluid
moves down the pipe, the more surface it is exposed to and, the
more surface it is exposed to, the more its flow is slowed by
friction. At some point from the origin, the accumulated friction
has removed so much energy from the fluid flow that it ceases to be
turbulent. This happens when, the Reynolds number falls below a
value of about 2000, and then the flow is said to be "laminar."
[0032] Laminar flow is not desirable in cell cultures because in
such conditions the cells have a tendency to aggregate, either by
sinking or swimming. Turbulent flow prevents such aggregations.
Imagine, for example, how sand particles would rapidly sink to the
bottom in a still pond, but would not do so in a large breaking
wave or a rapidly moving stream.
[0033] In summary, then, turbulent flow is maintained by avoiding
very low fluid velocities, very small characteristic lengths, and
very long channels. The characteristic length for horizontal Closed
Systems such as flat-plate reactors or horizontal tubes is the
depth of the culture, which, as explained previously, has a
practical upper limit of about 20 cm. One can create turbulent flow
in a flat-plate reactor or a horizontal tube with any number of
devices such as pumps or airlifts. However, with increasing
distance from the origin of the flow, turbulent energy is lost to
friction such that, at some finite distance flow becomes laminar.
In laminar flow conditions the cells of most photosynthetic
microbes will sink to the bottom of the reactor. This is
undesirable for many reasons, not the least of which is that
harvesting the cells becomes problematical. One solution is to
provide more turbulent energy at the source, but this is acceptable
only to an upper limit where mechanical shear damages the cells
themselves. Yet another solution is to provide multiple pumps, for
example, throughout the reactor, but this approach introduces
additional complexities of both construction and operation.
[0034] As a matter of practice, vertical Closed Systems are limited
to a capacity of less than about 1,000 liters, and horizontal
Closed Systems appear to be limited to capacities less than about
50,000 liters. For the purpose of large-scale cultivation of
photosynthetic microbes, Closed Systems are much more costly and
complex to construct and operate than Open Systems. This is because
each independent system requires its own independent
infrastructure: a set of devices or mechanisms for providing
turbulent mixing, introduction and removal of medium, and
monitoring and control of variables such as pH and temperature. To
cover a given area of land with Closed Systems requires at least 10
times more infrastructure than covering the same area of land with
Open Systems, rendering Closed System cultivation much more
complicated.
[0035] In practice, every cultivation system for photosynthetic
microbes involves a coupling of both Open Systems and Closed
Systems at some scale. All cultivation systems, regardless of
scale, ultimately depend for their original inoculum of cells on
culture collections routinely maintained around the world. All
culture collections exclusively maintain their cell cultures in
Petri dishes, test tubes, or sterilized flasks--all of which are,
strictly speaking, Closed Systems. Even large-scale production
systems that might be considered to consist "purely" of Open
Systems must rely ultimately on a Closed System to supply the
original inoculum.
[0036] The main technical conundrum for the production of
photosynthetic microbes is that Open System technology has advanced
to a large scale that is economical and relatively easy to operate,
but cannot provide sustainable production of desired microbes. By
contrast, Closed Systems do provide sustainable production of
desired microbes, but even at their largest scale they are costly
and complicated to operate.
[0037] Thus, there is a need for a production method that provides
for sustainable production by reducing the potential for
contamination and yet does not substantially increase the
complexity or cost of construction or operation.
[0038] It is therefore an object of this invention to provide an
effective method for sustainable production of photosynthetic
microbes at large scales that may be easily constructed and does
not increase the complexity or cost of construction or
operation.
[0039] It is a still further object of this invention to provide a
method of production that is especially suited to optimizing the
production of oils and other useful products from photosynthetic
microbes. Oils and other useful products may then be extracted and
purified from the aggregate biomass by means of a variety of
chemical methods.
SUMMARY OF THE INVENTION
[0040] These and other objects are achieved by a two-stage,
continuous-batch production methodology, wherein the first stage of
continuous production is accomplished in Closed Systems and the
second stage of batch production is accomplished in Open Systems.
First, one must select (including creating by, for example, genetic
modification) a microbe that is capable of growing at a rate of at
least one doubling every 16 hours, when supplied with sufficient
carbon dioxide, provided that light and nutrients are adequate.
Such genetic modification is disclosed, for example, in "Transgenic
microalgae as green cell-factories," by R. Leon-Banares, D.
Gonzalez-Ballester, A. Galvan and E. Fernandez (in Trends in
Biotechnology, Volume 22(1), pp. 45-52, 2004), which is
incorporated herein by reference. For example, it is presently
preferred to practice this invention with the following species
and/or strains: (i) Tetraselmis suecica, "TETRA1" strain in the
University of Hawaii Culture Collection; Isochrysis galbana,
"ISOCH1" strain in the University of Hawaii Culture Collection;
(iii) Phaeodactylum tricornutum, either strain "PHAEO1" or "PHAEO2"
in the University of Hawaii Culture Collection; or (iv)
Nannochloropsis sp., in particular the A. Sukenik strain used by
Fabregas et al. (2004), reported in "The cell composition of
Nannochloropsis sp. changes under different irradiances in
semicontinuous culture," World Journal of Microbiology and
Biotechnology, Vol. 20, pp. 31-35. The University of Hawaii Culture
Collection includes the entire culture collection of several
hundred species and strains amassed in the 1980s and 1990s by the
U.S. Natural Renewable Energy Laboratory (NREL), specifically for
the purpose of producing biofuels from photosynthetic microbes.
Additional species that might also be used include Dunaliella
primolecta and Nitzschia closterium. In particular, Closed Systems
should comprise no more than 20% of the Total Land Area of
cultures, i.e. the total area occupied by the Closed System Area
plus the Open System Area. Furthermore, given that every species of
photosynthetic microbe attains a maximum biomass per unit Lighted
Area under a given set of environmental conditions (the "carrying
capacity"), the amount of biomass provided by the Closed Systems to
initiate or "inoculate" any Open System culture should be equal to
more than 5% of the carrying capacity of the Open System. To
maximize the carrying capacity of the Open System cultures, they
should be provided with sufficient nutrients so that light, not
nutrients, limits the carrying capacity. Additionally, no batch
culture in any Open System should be allowed to persist for a
period (the "residence time") of more than 5 days.
[0041] The limitation of 20% of the Total Land Area occupied by the
Closed System Area assures that the complexity of construction and
operation of the entire production facility is minimized. The
provisions for the minimum biomass inoculum and the maximum
residence time assure that the risks of contamination of the Open
System cultures by undesirable species are so reduced as to be
inconsequential. This process may be used for any species of
photosynthetic microbe that meets the growth rate requirements
stated above.
DESCRIPTION OF A PREFERRED EMBODIMENT
[0042] It is preferred that the Total Land Area in active
cultivation at any production facility be comprised of no more than
20% Closed System Area and no less than 80% Open System Area. The
calculation of the Open System Area for this purpose means only the
Lighted Area of culture medium in the Open Systems, assuming that
all Open Systems in the production facility contain culture medium.
The calculation of the Closed System Area, however, includes both
the "Plan Area" covered by the reactors and also any "Inert Area"
between adjacent reactor vessels. Imagine, for example, an area of
land occupied by a series of horizontal tubular Closed Systems. The
Plan Area will be covered by the reactors themselves (e.g. a 10
foot long tube, 1 foot in diameter, covers an area of 10 square
feet), and is equal to the Lighted Area of the culture medium, but
there may be additional Inert Area between adjacent tubes that is
not covered by reactors. The entire area of land required by the
Closed Systems, i.e the Closed System Area, is the Plan Area plus
the Inert Area. By analogy, the entire area of this page is
required for writing, but the writing itself occupies only a
fraction of the total area on the page.
[0043] The provision for 20% Closed System Area and 80% Open System
Area assures greater efficiency because the overall complexity of
construction and operation of any production facility is
substantially reduced by comparison to a facility that would be
comprised entirely of Closed Systems.
[0044] It is preferred that (1) the amount of biomass provided by
the Closed Systems to inoculate the Open Systems should be equal to
more than 5% of the carrying capacity of the aggregate Open
Systems; (2) the growth rate of the species being cultivated is
greater than approximately one and a half doublings per day (i.e.
cell biomass doubles about every 16 hours); and that (3) no culture
be maintained in any Open System for a period of more than 5 days.
The combination of these three limitations assures that, under any
circumstances, the culture should attain a biomass of the desired
microbe that is equal to at least approximately 90% of the carrying
capacity in 5 days or less. This is important for several reasons.
First, a culture that is inoculated at a relatively high cell
concentration (i.e. greater than 5% of carrying capacity) will
dominate the medium compared to any unwanted cells that may have
inadvertently been introduced. Second, because most species grow at
rates substantially less than 1 doubling every 16 hours (1.5
doublings per day), a species that is capable of growing this
rapidly will outpace most potential competitors. Third, the
combination of the large inoculum (greater than 5% of carrying
capacity) and high growth rate (greater than 1 doubling every 16
hours) assures that, within 5 days, the total biomass will be very
near carrying capacity. These conditions are important to (1)
reducing the risk of contamination, and (2) promoting the
production of total biomass or the biosynthesis or production of
oil. First, a potential contaminant would have to have a large
inoculum and would have to grow more rapidly than the desired
species to dominate the culture medium within 5 days. Second, oil
production in particular is favored in cultures that are near
carrying capacity because resources become limiting to growth once
the culture passes 50% of carrying capacity. By limiting resources
favorable to growth, one generally stimulates the biosynthesis of
oil.
[0045] It is preferred that the cell cultures are supplied with
gaseous carbon dioxide in a manner that permits the carbon dioxide
to dissolve in the medium. This method not only provides a constant
source of the carbon needed for growth, but also has the effect of
maintaining more or less constant pH of the medium, without which
the pH would tend to increase as carbon dioxide is removed, leading
to unfavorable conditions for growth. Optimally, the carbon dioxide
for this process is provided by point source emissions from
resource extraction processes (such as drilling for oil or gas), or
from industrial manufacturing processes or fossil-fuel burning
power plants--all of which involve a waste stream of gas that is
rich in carbon dioxide. The process described here avoids emission
of the waste carbon dioxide into the atmosphere and instead
converts it into potentially useful biomass.
[0046] One could improve upon the preferred conditions. Optimally,
one could closely approach carrying capacity in the aggregate Open
Systems within one day, which would further reduce the potential
for contamination. This could be achieved by using an initial
inoculum of 15% of aggregate Open System carrying capacity for a
species with a growth rate of more than 6 doublings per day
(equivalent to 1 doubling of cell biomass about every 4 hours). In
this example, imagine that the species grows at the same rate in
both Closed Systems and Open Systems, and that the species also
attains the same concentration of biomass per unit area in both
Closed Systems and Open Systems. Now, if Closed Systems occupy 20%
of the Total Land Area, then a 15% inoculum for the Open Systems
would be obtained by removing 75% of the Closed System culture
(i.e. 75%.times.20%=15%). Using a 15% inoculum in the Open Systems,
the cell biomass would double to 30% in the first 4 hours, to 60%
in the next 4 hours, and in less than the next 4 hours could
achieve 100% (i.e. its carrying capacity). The Closed Systems will
be restored to their pre-inoculation biomass in about the same
amount of time. The Closed Systems contain only 25% of their
original biomass after inoculum is removed; in the first 4 hours
this doubles to 50% of the original biomass, and in the second 4
hours, it doubles again, this time to 100% of the original biomass.
In this example, the carrying capacity of the Open Systems is
achieved within one day and, within the same day, the biomass of
the Closed Systems is restored to its initial value. The highest
exponential growth rate recorded for photosynthetic microbes is
less than about 8 doublings per day (equivalent to 1 doubling about
every 3 hours), even better results might be achieved in less time
with a greater amount of initial inoculum.
[0047] The preferred method, using a species with a growth rate
greater than about 1.5 doublings per day, inoculating the Open
System with a biomass of at least 5% of carrying capacity, and
harvesting the culture no later than 5 days after inoculation,
assures a relatively high oil content. It also assures that the
Open System culture will not be significantly contaminated, since
decades of experience show that Open System cultures usually take
more than 5 days--usually several weeks--to become contaminated by
undesired species.
[0048] To practice the preferred method it is necessary to first
determine the carrying capacity of the Open System. Carrying
capacity for most species provided with excess nutrients in a
turbulent flow environment is generally in the range of about 100
to 500 grams dry weight per square meter of Lighted Area.
Variations in this range will depend primarily upon light
intensity, more specifically the total daily light energy of the
sun, as the limiting factor. For example, the total "irradiance"
(light energy of the sun) on a sunny day is about twice as much as
it is on a cloudy day. The corresponding difference in carrying
capacity would be about the same, i.e. about a factor of two.
Furthermore, the average irradiance is greatest in the tropics, and
decreases at higher latitudes. For example, average irradiance in
tropical Honolulu is about 40% greater than in New York, and about
2 times greater than in northern Alaska. Accordingly, the average
carrying capacity in Alaska would only be half of that in Hawaii.
The greatest irradiance of the year occurs on the longest day of
the year. North or south of the tropics, this happens on the first
day of summer (the summer solstice). At the equator, maximum daily
irradiance happens twice a year, when the sun passes directly
overhead on the first day of spring (the spring equinox) and once
again on the first day of fall (the fall equinox). Elsewhere in the
tropics, the maximum daily irradiance also happens twice a year,
somewhere between the spring and fall equinoxes.
[0049] The carrying capacity of a system for a desired species of
photosynthetic microbe should be determined empirically as follows.
If Open Systems in the production facility have different
dimensions, then the calculation of carrying capacity should be
determined for each set of systems having different dimensions.
Preferably, the determination of carrying capacity should be
carried out within about 15 days of the time when daily irradiance
is at its annual maximum. The Open System should be inoculated with
a volume of culture provided by the Closed Systems, and the Open
System filled approximately to standard operating capacity with
nutrient medium. Macronutrient concentrations (i.e. inorganic
nitrogen and phosphorus) should be provided in excess, defined by
demonstrating that once the biomass in the Open System has reached
its maximum, macronutrients will still be present in measurable
concentrations (the microbes won't have eaten all the food). After
inoculation, measurements of the biomass of the Open System should
be made at least several times per day until such time as the
biomass does not increase. The biomass at which the increase ceases
is the carrying capacity. The determination should be repeated at
least several times during the specified period, and the average of
these determinations calculated to define the maximum carrying
capacity.
[0050] The present invention has been disclosed with respect to the
presently preferred embodiments described herein, but those skilled
in the art will appreciate that there may be other embodiments that
fall within the sprit and the scope of the invention. Thus the
invention is not limited by what is described in the specification,
but is only limited by the claims. For example, this invention can
be practiced with different types of Open Systems and Closed
Systems. Furthermore, this invention can be practiced with
photosynthetic microbes with widely varying growth rates and
carrying capacities, and is not limited to those species being
cultivated for their oil content, but can also include species that
accumulate other products of value in batch cultures, whether or
not they have attained a maximum rate of biomass production or they
have stopped growing.
INDUSTRIAL APPLICABILITY
[0051] This invention can be used whenever it is desired to produce
any specific species of photosynthetic microbe in an Open System
while avoiding substantial contamination by undesired species. The
biomass thus produced from photosynthetic microbes can then be used
as a renewable feedstock to manufacture products that now rely
almost exclusively on feedstocks derived from fossil deposits of
carbon, namely coal, oil and gas. For example, the oil fraction of
photosynthetic microbes may be extracted and chemically converted
to transportation fuel, such as biodiesel, or to lubricants.
Alternatively, the biomass feedstock may be used in a process such
as fluidized bed gasification, pyrolysis, or entrained flow
gasification, from which the resulting material may be used in turn
to manufacture transportation fuels such as biodiesel or dimethyl
ether, or bulk chemicals such as methanol or mixed alcohols or, for
that matter, any product that uses fossil fuels as a starting
material or feedstock. Additionally, carbon-containing waste
products from any of the above processes could yield further energy
by methods such as anaerobic digestion, which produces methane
(natural gas), or co-firing with other combustible fuels in a power
plant to produce electricity.
* * * * *