U.S. patent application number 15/821307 was filed with the patent office on 2018-04-19 for bioreactor array and methods of combinatorial testing.
The applicant listed for this patent is HELIAE DEVELOPMENT LLC. Invention is credited to Thomas Adame, Eneko Ganuza Taberna, Michael LaMont, Steven Pflucker, Shan Qin, Anna Lee Tonkovich.
Application Number | 20180105783 15/821307 |
Document ID | / |
Family ID | 50336494 |
Filed Date | 2018-04-19 |
United States Patent
Application |
20180105783 |
Kind Code |
A1 |
Tonkovich; Anna Lee ; et
al. |
April 19, 2018 |
BIOREACTOR ARRAY AND METHODS OF COMBINATORIAL TESTING
Abstract
Methods and apparatus for culturing microorganisms are
described, including culturing in mixotrophic culture conditions. A
bioreactor array with multiple culture vessels with independently
controllable inputs is used to culture similar cultures of
microorganisms in which at least one parameter differs from other
culture vessels in the bioreactor array.
Inventors: |
Tonkovich; Anna Lee;
(Gilbert, AZ) ; Adame; Thomas; (Chandler, AZ)
; Qin; Shan; (Gilbert, AZ) ; Ganuza Taberna;
Eneko; (Tempe, AZ) ; LaMont; Michael;
(Gilbert, AZ) ; Pflucker; Steven; (Mesa,
AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HELIAE DEVELOPMENT LLC |
Gilbert |
AZ |
US |
|
|
Family ID: |
50336494 |
Appl. No.: |
15/821307 |
Filed: |
November 22, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14805267 |
Jul 21, 2015 |
9856447 |
|
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15821307 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 23/38 20130101;
C12M 35/00 20130101; C12M 23/44 20130101; C12M 41/36 20130101; C12M
23/58 20130101; C12M 41/18 20130101; C12M 41/12 20130101; C12M
29/20 20130101; C12M 41/00 20130101; C12M 41/26 20130101; C12M
41/48 20130101; C12M 21/02 20130101; C12M 29/00 20130101; C12M
33/00 20130101; C12M 41/34 20130101; C12M 41/06 20130101; C12M
41/32 20130101; C12M 23/22 20130101 |
International
Class: |
C12M 1/00 20060101
C12M001/00; C12M 1/36 20060101 C12M001/36; C12M 1/34 20060101
C12M001/34; C12M 1/02 20060101 C12M001/02; C12M 1/42 20060101
C12M001/42; C12M 1/26 20060101 C12M001/26; C12M 3/00 20060101
C12M003/00 |
Claims
1-20. (canceled)
21. A method for evaluating the feasibility of large scale
microalgae culture, comprising: a. providing a plurality of
microalgae cultures in an array of closed culturing vessels,
wherein (1) each vessel of the array comprises (a) a plurality of
sensors selected from the group consisting of a pH sensor, a
temperature sensor, a light sensor, a dissolved oxygen sensor, a
dissolved carbon dioxide sensor, and an optical density sensor and
(b) a plurality of controls selected from controls for pH, light,
gas levels, media levels, temperature, culture agitation, and
culture harvesting; (b) the culturing vessels are linked to one or
more shared reservoirs for controllably adding culture media, gas,
or both to the vessels; and (c) each culture comprises at least 0.5
grams of microalgae in a volume of at least 500 ml of an aqueous
culture medium; b. independently subjecting each microalgae culture
to one or more related test conditions wherein at least a plurality
of the cultures is subjected to different degrees of at least one
test condition; and c. evaluating the impact of the test conditions
on the cultures to determine the effect of the test conditions on
the microalgae and assessing the feasibility of performing large
scale commercial microalgae culture under one or more production
conditions related to the test conditions from the results of the
evaluation.
22. The method of claim 21, wherein the method comprises
contemporaneously culturing a plurality of test cultures and a
plurality of control cultures in the array.
23. The method of claim 22, wherein the method comprises
contemporaneously culturing a plurality of cultures under the same
conditions such that this plurality of cultures serve as
repetitions in an experiment.
23. The method of claim 22, wherein the method comprises
contemporaneously culturing at least three control cultures and at
least three test cultures.
24. The method of claim 23, wherein the method comprises
contemporaneously culturing a plurality of cultures under the same
conditions such that this plurality of cultures serve as
repetitions in an experiment.
25. The method of claim 23, wherein the method comprises
contemporaneously testing three or more different conditions in the
array by culturing three or more cultures wherein each culture of
the three or more cultures is cultured under a different
condition.
26. The method of claim 24, wherein the method comprises culturing
multiple cultures for each of the three or more different
conditions being tested in the array, wherein each culture being
tested for a condition is cultured under different levels of the
condition.
27. The method of claim 26, wherein the different conditions
comprise culturing the microalgae in the presence of different
chemicals.
28. The method of claim 26, wherein the method comprises
contemporaneously testing five or more different conditions in the
array.
29. The method of claim 21, wherein the method comprises adding
gas, media, or both, to a plurality of the cultures during the
culture process from a shared media reservoir, gas reservoir, or
both.
30. The method of claim 29, wherein the method comprises supplying
gas, supplying media, or both, to one or more vessels by passing
the gas, media, or both through a lid that encloses the top of the
vessel.
31. The method of claim 25, wherein the method comprises adding
gas, media, or both, to a plurality of the cultures during the
culture process from a shared media reservoir, gas reservoir, or
both.
32. The method of claim 31, wherein the method comprises supplying
gas, supplying media, or both, to one or more vessels by passing
the gas, media, or both through a lid that encloses the top of the
vessel.
33. A system for testing the feasibility of large scale culture of
microalgae under different conditions comprising: (a) an array of
at least eight substantially identical culturing vessels which are
suitable for culturing microalgae, wherein each vessel is capable
of containing a culture of at least 500 mL in volume and comprises
(i) a plurality of sensors selected from the group consisting of a
pH sensor, a temperature sensor, a light sensor, a dissolved oxygen
sensor, a dissolved carbon dioxide sensor, and an optical density
sensor and (ii) a plurality of controls selected from controls for
pH, light, gas levels, media levels, temperature, culture
agitation, and culture harvesting; (b) a shared gas reservoir and
delivery system, which is connected to each of the culturing
vessels in the array and is capable of independently and
controllably adding gas to each of the vessels; (c) a shared media
reservoir and delivery system, which is connected to each of the
culturing vessels in the array and is capable of independently and
controllably adding media to each of the vessels; and (d) a
programmable logic controller that controls at least a plurality of
the controls and receives data from at least a plurality of the
sensors in each of the cultures.
34. The system of claim 33, wherein more than one of the culture
vessels is light transmissive.
35. The system of claim 34, wherein each of the culture vessels
comprises a lid through which a gas supply inlet and a nutrient
supply inlet passes.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. patent
application Ser. No. 14/805,267, filed Jul. 21, 2015, entitled
Bioreactor Array and Methods of Combinatorial Testing, and PCT
Application No. PCT/US2014/016462, filed Feb. 14, 2014, entitled
Bioreactor Array and Methods of Combinatorial Testing, U.S.
Provisional Application No. 61/850,623, filed Feb. 19, 2013,
entitled Photobioreactor Array and Mixotrophic Culture, and U.S.
Provisional Application No. 61/917,423, filed Dec. 18, 2013,
entitled Photobioreactor Array and Mixotrophic Culture, the entire
contents of which are hereby incorporated by reference.
BACKGROUND
[0002] Photosynthetic organisms have been cultured to produce
chemicals and biologicals of interests such as fatty acids,
proteins, hydrogen gas, pigments, carbohydrates, sugars, and
vitamins for use in food, feed, pharmaceuticals, nutraceuticals,
fuels, and other products.
[0003] Of particular interest in recent years has been the use of
microorganisms, such as microalgae and photosynthetic bacteria
cultures, themselves or extracts derived from the microorganism. A
number of microalgal metabolites have commercial interest as
chemical compounds and have been so produced. Many have attempted
to grow microalgae, typically in open ponds, long tubes, and bags
with tubes between them. Others have attempted growing microalgae
in enclosed tank systems, but each type of system has encountered
difficulties related to control and optimization of the microalgae
culture. Heterotrophic microorganisms, such as microalgae and
bacteria, have also been cultured in traditional stainless steel
fermenters to produce for similar chemicals and biologicals for
commercial products in the same fields.
[0004] In an attempt to enhance production of a microalgae culture,
a parameter of the biocultivation process has been varied in an
attempt to find the optimum range for that parameter.
[0005] However, it is known that multiple parameters interact with
each other within a culture to give complex relationships regarding
optimal ranges for each parameter. Even a simple component, such as
the culture medium under auxotrophic conditions containing no more
than salts, lacks simplistic optimization. For example, Pandey et
al, Journal of Algal Biomass Utilization, 1(3) p. 70-81 (2010),
reports very different yields using differing proportions of salts
in their culturing medium even with all other culture parameters
remaining unchanged. Within the article, it is noteworthy that no
single salt concentration had an optimal range that controlled
yield. Even different conventional culture media resulted in
doubling the yield. The media compared were all previously
published for Spirulina and previously optimized for Spirulina
growth, yet when compared side-by-side, dramatically different
yields resulted.
[0006] Given the potential market, the need for a large-scale
microalgae growth system is evident; yet, the prior art systems
have had little optimization before being built. Different
bioreactors present different problems and are optimized
differently around the parameters which are not changeable. Open
ponds suffer from problems with contamination (e.g., organics,
bacteria, fungi), changing conditions throughout the day and night
(e.g., temperature, sunlight, wind), and other suboptimal
cultivation conditions. Many bioreactors have difficulties with
sufficient light reaching the microalgae for photosynthetic
activity and in controlling the conditions (e.g., temperature, pH,
nitrate levels). A system relying solely on ambient light as an
energy source for the microalgae is susceptible to fluctuation with
seasons and even clouds, which may vary random and non-reproducible
manner making comparison between different runs difficult or
impossible to interpret even when the same bioreactor is used.
[0007] Therefore, there is a need in the art for a system and
method of optimizing culturing parameters to increase the
efficiency of microalgae cultivation.
SUMMARY
[0008] The present invention provides a method and apparatus for
cultivating microorganisms in a liquid or aqueous culture medium
with control over the culturing parameters.
[0009] In one embodiment, a bioreactor array may comprise a
plurality of culture vessels configured to contain an aqueous
culture of microorganisms in an interior volume, each culturing
vessel comprising an independently controllable: gas supply,
nutrient supply, heat exchanger, harvesting mechanism, and light
supply; at least one sensor configured to measure at least one
culturing parameter of each culture vessel; and a programmable
logic controller. In some embodiments the culture vessel may have a
volume of 500 to 1000 ml. In some embodiments, the culture vessel
may be transparent. In some embodiments, the culture vessel may
comprise at least one opaque section.
[0010] In some embodiments, the gas supply may comprise at least
one gas selected from the group consisting of air, carbon dioxide,
oxygen, and nitrogen. In some embodiments, the nutrient supply may
comprise an organic carbon supply. In some embodiments, the
harvesting system may comprise an overflow system configured to
passively remove at least part of the aqueous culture volume at a
culture volume less than a total volume of the culture vessel. In
some embodiments, the at least one sensor may be selected from the
group consisting of: pH sensor, temperature sensors, light sensors,
dissolved oxygen sensors, dissolved carbon dioxide sensor, and
optical density sensor. In some embodiments, the light supply may
comprise a lighting device disposed outside of the culture vessel.
In some embodiments, the light supply may comprise a lighting
device disposed in the interior volume of the culture vessel.
[0011] In another embodiment, a method of culturing microorganisms
may comprise providing culture of microorganisms in an aqueous
culture medium in a plurality of culture vessels; independently
controlling the supply to each culture vessel at least one from the
group consisting of: light, at least one gas, at least one
nutrient, and heat exchange; and wherein each culture vessel
contains a culture of the same microorganisms cultured with
different parameters selected from the group consisting of
temperature, pH, amount of light, intensity of light, wavelengths
of light, light photoperiod, light/dark cycle, concentrations of
gases, and agitation from gas supply. In some embodiments, the at
least one nutrient may comprise organic carbon.
[0012] In some embodiments, the method may further comprise
harvesting at least part of the aqueous culture from the culture
volume. In some embodiments, the different parameters may further
comprise harvest rates. In some embodiments, the culture vessel may
be supplied with light and organic carbon. In some embodiments, the
culture may be supplied with organic carbon but no light.
[0013] In some embodiments, providing the culture of microorganisms
in the aqueous culture medium in each culture vessel may comprise
0.5-1.5 grams of biomass. In some embodiments, providing the
culture of microorganisms in the aqueous culture medium in each
culture vessel comprises 500 to 1,000 ml of culture volume. In some
embodiments, the method may further comprise monitoring the
parameters of the culture of microorganisms in the plurality of
culture vessels with at least one sensors and a programmable logic
controller. In some embodiments, the supply of light may comprise
at least two independently controllable light emitting diodes
providing different wavelengths of light.
[0014] The current invention provides a method for strain
conditioning, adaptation and selection. The method may be performed
in a plurality of bioreactors where at least one parameter
stressing the microorganism culture differs between bioreactors.
Alternatively, the inventive reactors may be used to evaluate
growth parameters or treatments (chemical or biological) to control
or reduce contamination in microorganism cultures.
[0015] In another embodiments, the bioreactor array may comprise: a
plurality of culture vessels configured to contain an aqueous
culture of microorganisms in an interior volume, each culturing
vessel comprising means for controlling the gas composition,
nutrient composition, temperature, light exposure, and harvest of
the aqueous culture of microorganisms; and means for measuring at
least one culturing parameter of each culture vessel. In some
embodiments, the bioreactor array may further comprise computer
automated means for controlling at least one of the gas
composition, nutrient composition, temperature, light exposure, and
harvest of the aqueous culture of microorganisms in response to the
at least one culturing parameters. In some embodiments the at least
one culturing parameter may comprise at least one from the group
consisting of: nutrient concentration, temperature, pH, amount of
light, intensity of light, wavelengths of light, light photoperiod,
light/dark cycle, concentrations of gases, and agitation from gas
supply
DETAILED DESCRIPTION
[0016] While not wishing to be bound by any particularly theory,
the present invention is believed to determine desirable effects on
microorganisms, including photosynthetic microorganisms, and/or
products as the result of the metabolism of microorganisms
used.
[0017] For the purposes of this specification the term
"photosynthetic microorganism" is intended to cover any
phototrophic or mixotrophic or microorganism that is capable of
utilizing light as a source of energy through photosynthesis. The
photosynthesis need not be directly involved in producing the
desired result. The photosynthesis need not even occur provided
that an alternative energy source is provided. All organisms that
utilize light for photosynthesis, of which phototrophic and
mixotrophic species of microorganism are included.
[0018] The term "microorganism" refers to microscopic organisms
such as microalgae and cyanobacteria. Microalgae include
microscopic multi-cellular plants (e.g. duckweed), photosynthetic
microorganisms, heterotrophic microorganisms, diatoms,
dinoflagelattes, and unicellular algae.
[0019] The terms "microbiological culture", "microbial culture", or
"microorganism culture" refer to a method or system for multiplying
microorganisms through reproduction in a predetermined culture
medium, including under controlled laboratory conditions.
Microbiological cultures, microbial cultures, and microorganism
cultures are used to multiply the organism, to determine the type
of organism, or the abundance of the organism in the sample being
tested. In liquid culture medium, the term microbiological,
microbial, or microorganism culture generally refers to the entire
liquid medium and the microorganisms in the liquid medium
regardless of the vessel in which the culture resides. A liquid
medium is often referred to as "media", "culture medium", or
"culture media". The act of culturing is generally referred to as
"culturing microorganisms" when emphasis is on plural
microorganisms. The act of culturing is generally referred to as
"culturing a microorganism" when importance is placed on a species
or genus of microorganism. Microorganism culture is used
synonymously with culture of microorganisms.
[0020] The terms "phototrophic", "phototrophy", "photoautotrophy",
"photoautotrophic", and "autotroph" refer to culture conditions in
which light and inorganic carbon (e.g., carbon dioxide, carbonate,
bi-carbonate) may be applied to a culture of microorganisms.
Microorganisms capable of growing in phototrophic conditions may
use light as an energy source and inorganic carbon (e.g., carbon
dioxide) as a carbon source. A microorganism in phototrophic
conditions may produce oxygen.
[0021] The terms "mixotrophic" and "mixotrophy" refer to culture
conditions in which light, organic carbon, and inorganic carbon
(e.g., carbon dioxide, carbonate, bi-carbonate) may be applied to a
culture of microorganisms. Microorganisms capable of growing in
mixotrophic conditions have the metabolic profile of both
phototrophic and heterotrophic microorganisms, and may use both
light and organic carbon as energy sources, as well as both
inorganic carbon and organic carbon as carbon sources. A
mixotrophic microorganism may be using light, inorganic carbon, and
organic carbon through the phototrophic and heterotrophic
metabolisms simultaneously or may switch between the utilization of
each metabolism. A microorganism in mixotrophic culture conditions
may be a net oxygen or carbon dioxide producer depending on the
energy source and carbon source utilized by the microorganism.
Microorganisms capable of mixotrophic growth comprise
microorganisms with the natural metabolism and ability to grow in
mixotrophic conditions, as well as microorganisms which obtain the
metabolism and ability through modification of cells by way of
methods such as mutagenesis or genetic engineering.
[0022] The terms "heterotrophic" and "heterotrophy" refer to
culture conditions in which organic carbon may be applied to a
culture of microorganisms in the absence of light. Microorganisms
capable of growing in heterotrophic conditions may use organic
carbon as both an energy source and as a carbon source. A
microorganism in heterotrophic conditions may produce carbon
dioxide.
[0023] The organic carbon sources suitable for growing a
microorganism mixotrophically may comprise: acetate, acetic acid,
ammonium linoleate, arabinose, arginine, aspartic acid, butyric
acid, cellulose, citric acid, ethanol, fructose, fatty acids,
galactose, glucose, glycerol, glycine, lactic acid, lactose, maleic
acid, maltose, mannose, methanol, molasses, peptone, plant based
hydrolyzate, proline, propionic acid, ribose, sacchrose, partial or
complete hydrolysates of starch, sucrose, tartaric, TCA-cycle
organic acids, thin stillage, urea, industrial waste solutions,
yeast extract, and combinations thereof. The organic carbon source
may comprise any single source, combination of sources, and
dilutions of single sources or combinations of sources.
[0024] Of particular use in the present invention are mixotrophic
microorganisms such as, but not limited to Agmenellum, Amphora,
Anabaena, Anacystis, Apistonema, Pleurochyrsis, Arthrospira
(Spirulina), Botryococcus, Brachiomonas, Chlamydomonas, Chlorella,
Chloroccum, Cruciplacolithus, Cylindrotheca, Coenochloris,
Cyanophora, Cyclotella, Dunaliella, Emiliania, Euglena,
Extubocellulus, Fragilaria, Galdieria, Goniotrichium,
Haematococcus, Halochlorella, Isochyrsis, Leptocylindrus,
Micractinium, Melosira, Monodus, Nostoc, Nannochloris,
Nannochloropsis, Navicula, Neospongiococcum, Nitzschia., Odontella,
Ochromonas, Ochrosphaera, Pavlova, Picochlorum, Phaeodactylum,
Pleurochyrsis, Porphyridium, Poteriochromonas, Prymnesium,
Rhodomonas, Scenedesmus, Skeletonema, Spumella, Stauroneis,
Stichococcus, Auxenochlorella, Cheatoceros, Neochloris, Ocromonas,
Porphiridium, Synechococcus, Synechocystis, Tetraselmis,
Thraustochytrids, Thalassiosira, and species thereof. Also included
may be more unclassified new microalgal genera, species, or strains
which may be poorly characterized or even newly discovered
microorganisms. While certain high yielding strains are
preferentially used, a considerable number of other organisms are
known and under the property conditions may also produce biomass
and various metabolites of interest. National Renewable Energy
Laboratory (NREL) has selected 300 species of microalgae, both
fresh water and salt water microalgae, including diatoms and green
microalgae. Other organizations have large depositories of
photosynthetic and non-photosynthetic microorganisms. Any of these
and others may be used in the present invention. Known literature
also describes a plurality of microorganisms capable of growth in
phototrophic and heterotrophic culture conditions which is also of
interest with this invention.
[0025] For the purposes of this specification the term "parameter"
refers to any feature of the culturing of the microorganism or
extraction or processing of the resulting culture. Examples may
comprise: bioreactor design; microorganism strain selection;
additional microorganism strain selection (if mixed culture);
selection of media components and concentration of each; dissolved
gases; gases in the atmosphere above the microorganism culture;
carbon dioxide, oxygen and other gas amounts and pressures in the
bioreactor; gas sparaging rate; gas bubble size; chemical carbon
source (if any); content of, timing of and rate of addition of
additional nutrients (e.g., organic carbon); fluid withdraw rate
(i.e., harvesting rate); culture mixing rate; cell withdraw rate;
initial cell concentration; maintenance of steady cell
concentration; pH; salinity; osmotic pressure; temperature; amount
of light; wavelength(s) of light; pulsing duration and rate of
light; length of light and dark cycles; rate of increasing and
decreasing light intensity during the lighting phase; selection of
a chemical enhancing product production or its amount or timing of
its addition; selection of chemical or physical inhibition of
contaminating microorganisms (amount and timing also); manner of
and rate of removal of wastes or contaminants; choice of added
contaminant or proxy to mimic natural contamination; type of
culture operation (e.g., batch, semi-batch, fed-batch, continuous);
rate and timing for removal of secreted product(s); longevity of
the culture; co-cultivation strategies and strain interactions; and
timing of a change in one or more parameter in response to a set
schedule or in response to a change in condition of one or more
other condition.
[0026] The term "axenic" describes a culture of an organism that is
entirely free of all other "contaminating" organisms (i.e.,
organisms that are detrimental to the health of the microalgae or
cyanobacteria culture). Throughout the specification, axenic refers
to a culture that when inoculated in an agar plate with bacterial
basal medium, does not form any colonies other than the
microorganism of interest. Axenic describes cultures not
contaminated by or associated with any other living organisms such
as but not limited to bacteria, cyanobacteria, microalgae and/or
fungi. Axenic is usually used in reference to pure cultures of
microorganisms that are completely free of the presence of other
different organisms. An axenic culture of microalgae or
cyanobacteria is completely free from other different
organisms.
[0027] Should one seek to optimize multiple parameters, the
combinations indicate one will generate a large set of test data of
different culture parameters. This is particularly challenging for
mixotrophic cultures with many additional complicating factors.
This is impractical to attempt in a mass production system as it
may take years of testing to optimize the parameters. Even then,
because some of the apparatus and photosynthetic organisms drift
over time, the comparison is far from perfect. By contrast, the
present invention mimics mass production in a more manageable way
which allows for easier evaluation of parameter combinations.
[0028] The present invention may isolate key parameters and mimic
the conditions in a mass production system to allow optimization
for different cultures of microorganisms. In mixotrophic culture
the impact of light is less important than the contribution of
growth energy and carbon from the organic carbon source. The units
in the described invention are well mixed which allows for a
volumetric reaction rate. For the mixotrophic reactions, growth is
dominated by volumetric reactions rather than areal reactions
resultant from solar input. Artificial light or solar light may be
supplemental, and the majority of growth may be resultant from the
organic carbon source.
[0029] Previous attempts to optimize parameters of a microorganism
culture have relied upon rather simple lab scale selections which
do not resemble real life situations, and thus the data produced
does not translate to improved cultures in commercial production
conditions. For example, traditionally, optimal microorganism
strains are typically selected by growing different strains in
multiwelled plates or separate small volume vessels and measuring
the results of either cell growth or product production. These
multiwelled plates are not well mixed and do not allow for a
feedback mechanism with the consumption of carbon during the
reaction given their small culture volume.
[0030] The control mechanisms for such a screening are different
from those used in large scale production (i.e., commercial
production). Further, this may not reflect actual results in mass
production in a large bioreactor or even an open pond where
parameters such as dissolved gases, depth of the culture, and
lighting conditions vary dramatically from the screening system.
The traditional selection using small volumes with poor mixing may
not choose a desired optimal microorganism strain or culture
parameters and may be more likely to select a strain optimized for
a batch system resembling the selection system rather than any
large scale continuous system.
[0031] During the operation of the array of vessels in the present
invention, numerous sensors may be present with continuous
monitoring of culture parameters. This allows for a thorough
determination of preferred parameters. For example, one set of
parameters may be ideal for the first few days which destroy the
culture on, for example, day seven. Without constant monitoring,
one would assume this set of parameters is unacceptable when
looking only at the final result, however the set of parameter may
be ideal provided that one make an adjustment on, for example, day
five or harvest the culture before day seven.
[0032] Another embodiment of the present invention is the
determination of the approximate cost and benefit of mass
production using a photosynthetic microorganism in the bioreactor
design stage before even building such a plant. Unlike conventional
laboratory systems, the present invention mimics actual large scale
production conditions, permitting extrapolation and translation of
useful data.
[0033] While considerable work has been conducted on phototrophic
microalgae including the optimization of parameters for growth, no
systems are commercially available to rapidly test mixotrophic
cultures of microalgae in parallel for rapid combinatorial
screening of strains, process conditions, media, growth factors,
organic carbon source, contamination vectors, and more. The number
of parameters affecting growth and photosynthetic microorganism
longevity are greater in mixotrophic systems than for phototrophic
systems with the inclusion of the complexity of non-light energy
sources, such as an organic carbon source, and the ensuing
challenges of contamination (e.g., bacteria, fungi, ciliates) that
may feed on the organic carbon source.
[0034] The apparatus used in the present invention may be fitted
with a wide selection of sensors. Preferred sensors may comprise:
pH, temperature, dissolved oxygen (DO), dissolved carbon dioxide,
cell density, concentrations of various chemicals in the medium,
amount of light, and wavelengths of light received. A sample of
culture medium may be withdrawn for testing or the testing may be
done on or inside the culture vessel itself. One example of a
continuous flowing sensor of numerous components in water is
exemplified by U.S. Pat. No. 8,102,518.
[0035] The apparatus may be operated in an axenic mode or may be
operated in a non-axenic mode. An advantage to a non-axenic mode is
the understanding of the impact of growth conditions on bacteria
both in terms of proliferation and individual strains of bacteria.
The apparatus may be used to test the response in microalgae and
contaminants to treatments applied for increasing microalgae
growth, longevity, or reduce contaminants.
[0036] While every individual vessel may have its own set of
sensors, it may be beneficial to have a single common sensor for
each type of parameter. For example, by having a line to collect
exhaust gases from each vessel separately, the same sensor can
provide repeated sequential measurements from each gas stream.
Likewise, for withdrawing a small sample of liquid and running it
by a single sensor for each type of parameter before returning the
sample to culture vessel. This avoids any difficulty or drift in
separate sensors.
[0037] The light may be provided to the cultures by any
conventional lighting source such as fluorescent bulbs, light
emitting diodes (LEDs), ambient room light, sunlight, etc. In some
embodiments, the light source may be a lighting device exterior to
the vessel. Reflected, refracted and filtered light may also be
used. In some embodiments, light may be provided to the culture
from inside the culture vessels by a submerged light source such as
fiber optics, LEDs or an LED strip or other lighting device
disposed within the interior volume of the culture vessel.
Controlling lighting parameters such as the intensity, amount,
duration, pulsing, light/dark cycle, wavelengths provided, etc.,
are particularly desirable and these parameters may desirably
change during the culturing process and in response to other
changing parameters.
[0038] In one embodiment, light may be only applied to a portion of
the vessel to evaluate the impact of reduced light, no light, high
light, or partial light on the growth and productivity of the
target microorganisms as well as unwanted species (i.e.,
contamination).
[0039] These and other parameters that are harmful to the
photosynthetic microorganism may be used if desired to enhance
product production or the desirability of it. For example, at the
end of the culture cycle, one may wish to dramatically increase the
amount of light to photobleach (i.e., stress) the microorganism to
aid in recovery of a product by autolysis or separation of the
product.
[0040] The types of products that can be produced include, whole
cells, extracts, lipids, proteins, pigments, hormones,
polysaccharides, and others. The amounts and proportions of each
are frequently altered by altering the process parameters. The
product may be an action also such as degradation of wastes or
catalytic biotransformation of one chemical to another.
[0041] Changing parameters during cultivation to have a desirable
effect on the photosynthetic microorganism's metabolism may be
preferred. While laboratory experimental examples are known of
adding a chemical to enhance or stop production or growth, they are
not typically done under conditions suitable for mass production
due to a lack of understanding of the interactions and effects. The
present invention would allow for a systematic evaluation of
interactions through combinatorial testing and reduce risk for
introducing chemicals or treatments at a mass cultivation
scale.
[0042] Supplies of gases to the culture vessels of the present
invention may be provided by a common line from a common reservoir.
Individual valves and meters may adjust one or more gasses before
being optionally mixed together and supplied to the cultures. A
bubbler or sparger at or near the bottom of the culture vessels may
be used to deliver the gases and provide mixing in the culture.
Laminar flow of bubbles may be used to provide mixing for shear
sensitive microorganisms. If desired, mixing baffles or a separate
active mixer of any conventional type may be added.
[0043] During the culturing, addition of any chemical and/or
withdraw (and optional recycle) of culture medium (with
photosynthetic microorganisms) may be performed as a parameter. The
culture may be run in batch, fed batch, semi continuous or fully
continuous methods.
[0044] A further embodiment is the use of a fed-batch mode of
operation whereby the organic carbon source may be slowly fed as
the organisms grow. The organic carbon source may be used to
control pH or may be added sparingly to disfavor the competitive
production of by-products, which may include bacteria or other
unwanted microalgae species. The organic carbon source may be
concentrated or dilute. The volume of the culture increases with
the addition of the organic carbon source. In some embodiments, the
culture may be periodically harvested or continuously harvested
with an overflow port that controls the volume to a preset level.
The use of the parallel culturing vessels may be used to
concurrently test the different modes of harvest as a parameter
that affects growth and longevity.
[0045] While exemplified by a clear vessel, the vessel may comprise
translucent or even opaque regions in part or in its entirety, with
at least one source for delivering light or feature allowing light
to be delivered. For example, it may be desirable to have steel
supporting structures in contact with the vessel or to have the
vessel in contact with a metal heat sink for heating or cooling the
vessel (i.e., heat exchange), either of which may block light from
reaching the culture volume.
[0046] While the cultures are discussed as producing a product, the
culture may also be used to degrade or remove undesirable
substances from the culture medium. This would be especially
desirable for optimizing the culture parameters to degrade
wastewater or thrive on waste mediums. The culture may also react
with or catalytically transform one feedstock into a more desired
product during either growth or steady state conditions in light or
dark.
[0047] Some or all of the bioreactor vessels may be sealed which
allows for pressurization and prevents outside contamination from
entering the vessel.
[0048] After each batch or periodically if continuous, the
bioreactor may be easily disassembled for ease of cleaning. The
array of vessels in the bioreactor may be individually removable
for cleaning, analysis, product recovery, or medium addition.
[0049] The present invention may also incorporate the use of
disposable or recyclable components that may comprise the use of a
plastic bag to serve as the reaction vessel. A bag reaction vessel
may be disposed after conducting a desired experiment. The bag may
be supported by a structure for frame to maintain the distance from
the culture volume to the light source.
[0050] For the purposes of this specification the term "clear"
refers to transparent or translucent to light, particularly
allowing the transmission of the light wavelengths utilized for
photosynthesis by the photosynthetic microorganism. It is
understood that even "clear" vessels will have some light
transmission loss that may range from about 1 to 40%. The desirable
wavelengths, such as photosynthetically active radiation (PAR)
light or light promoting production of a product, may vary somewhat
between different species of photosynthetic microalgae. A "clear"
material may be opaque or hinder other wavelengths of visible light
and other wavelengths of electromagnetic radiation. A "clear"
material is clear only in the sense of its optical properties and
only to an adequate degree for allowing light to pass through to
the photosynthetic microorganism.
[0051] In another embodiment of the present invention, the light
source may emit only certain wavelengths of light that have value
in promoting growth or production of desired product. LEDs of a
specific wavelength or wavelength enhanced LEDs emitting light in
the desired wavelengths may be used. The light source need not
exclusively emit desired wavelengths (e.g., PAR light) but rather
may be enriched for desired wavelengths in this embodiment of the
invention. A number of different electro luminescence devices known
per se may be used.
[0052] Also, an optical filter may be added between the light
source and the photosynthetic microorganism to block harmful
wavelengths or wavelengths which reduce production of the desired
product(s). Harmful wavelengths may even degrade the desired
product(s) or degrade an intermediate in the metabolic pathway of
the desired product(s). Certain harmful wavelengths may not be
harmful to the photosynthetic microorganism but rather encourage a
different metabolism away from maximum production of the desired
product(s). The optical filter may be a device, thin film,
particle, or simple compound, which reflects or adsorbs some of the
undesired wavelengths or even neutral wavelengths. This filter may
be outside or inside a culture vessel.
[0053] Nutrients required by the photosynthetic microorganism, e.g.
sodium nitrate and sodium phosphate or others, may be added
manually either in the solid form by premeasured manual addition or
may be added manually or automatically as a premeasured diluted
solution in water via the top of the vessel or elsewhere in a line.
These nutrients may also be added with the organic carbon media,
which may be added continuously or semi-continuously in response to
changing parameters, such as pH or volume and the like. The
nutrients may be pure ingredients, known mixtures or relatively raw
materials such as the wastewater from a food processing facility.
All added products to the system are preferably sterile to limit
the amount of contamination introduced into the culture vessel. The
addition of CO.sub.2 creates carbonic acid in water that will lower
the pH during the cycle time of a batch, therefore the pH may be
constantly monitored (by testing a sample or by a pH sensor located
in the vessel or a line of circulating liquid) and a buffer or an
alkaline material, such as sodium hydroxide or sodium bicarbonate
etc., may be added via the same technique as the nutrients to
control the pH at the desired level for optimum organism growth and
performance. When the bioreactor is open vented, there may be no
pressure other than the liquid head pressure in the vessel and the
vessel may be run at or close to the full volume level. The top of
the vessel may be used for adding nutrients, mounting pH
instrumentation, adding control chemicals, and periodic internal
cleanout of the reaction vessel and lines. Likewise for a closed
bioreactor, the top may also be used for the same features provided
that they are sealed with the vessel top.
[0054] A feature of the present invention is to have common
reservoirs of materials to add to the multiple culture vessels.
They may use a common line with manifolds and individual or group
valves or adjustable meters to deliver the same or differing
amounts to different vessels. This provides further control of the
amounts and concentrations added to different test vessels thereby
providing better comparisons when one or more parameters are
changed.
[0055] Another feature may be to have automated control of the
various parameters based on preset programs or feedback loops from
the sensors. Since many slightly different cultures of
photosynthetic microorganisms may be present, it may be impractical
to monitor all of them separately.
[0056] Additionally, data may be collected for storage and
comparison analysis. Having as many of the parameters controlled
along with common data collection makes for a better comparison. In
the present invention, a simple run of the bioreactor apparatus may
provide a considerable amount of comparable data for data mining by
conventional statistical analysis such as the statistical programs
from SAS Institute Inc. (100 SAS Campus Drive, Cary, N.C.
27513-2414).
[0057] The concentrations of the nutrient and pH control solutions
are carefully selected to minimize damaging or killing the
microorganisms, and to provide long term control during the growth
and product maturation phases (e.g., oil accumulation, pigment
accumulation) of the process. Redundant pH probes may be included
to provide easy switchover to a new probe when the recording probe
fails to operate. It is noted that a system that filters the
microorganisms and provides clean water to the pH probes could
extend the life of the probes in this service.
[0058] Contamination by undesired microorganisms may be reduced by
adding an inhibitory gas, such as ozone, chlorine dioxide, ethylene
oxide etc., in the headspace or by the CO.sub.2 sparger or a
separate gas sparger. If a specific contaminant is of particular
concern, an antibiotic, which may be less harmful to the desired
photosynthetic organism, may be added to the culture medium.
Likewise, the culture conditions may be modified to inhibit the
contaminant without excessive harm to the photosynthetic
microorganism.
[0059] A preferred embodiment of the present invention is a
bioreactor design for changing conditions while culturing
photosynthetic microorganisms. The bioreactor allows for a batch of
microalgae or other microorganism to undergo several days of both
continuous growth and an oil (or other metabolite) buildup phase of
production in batch or semi-batch conditions. Alternatively,
continuous production may be used. The culture conditions may
alternate between enhancing growth of the photosynthetic
microorganism and enhancing production of a metabolite, such as a
fatty acid or other desired compound(s). The culture may also be
designed to degrade unwanted or harmful compounds in the
medium.
[0060] The area outside the culture vessel may use an air
ventilation system to remove the heat produced by the light bulbs
and other equipment.
[0061] Computer controlled adjustable motors and valves may be
responsive to the culturing process conditions. Preset parameters
and changes in parameters may be effected by computer control, by
feedback loop, or manually by an operator. Redundant manual or
override controls may be present. Specific operations may comprise:
the addition of pH control agents, gases, liquids, nutrients;
removal of culture liquid or gases; adjusting the agitation,
recirculation, and gas introduction rates; and temperature control.
The operation may be run continuously and indefinitely to study the
long-term effects or to test for and optimize microorganism
longevity.
[0062] At the end of a cycle, or continuously, when microorganisms
are removed from the system, the desired product(s) may be
extracted from the microorganism in a separate downstream process
such as, but not limited to, solvent extraction, supercritical
fluid extraction, and cell disruption. The method of extraction and
recovery will depend on the particular product(s) targeted, and are
preferably done by a manner known per se.
Example 1: The Bioreactor Array
[0063] A set of identical 1 liter glass culture vessels having a
working culture volume of 500-800 ml and columns with dimensions of
4.57 cm inside diameter (ID).times.5.1 cm outside diameter
(OD).times.61.0 cm height (H) (1.8'' ID.times.2.0'' OD.times.24''
H). A rubber stopper is pressed into the open top of the column to
serve as a lid. Holes were cut into the rubber stopper to
accommodate a 0.762 OD.times.66.04 cm (0.3'' OD.times.26''H) glass
capillary tube for aeration, a 0.601 cm OD.times.30.48 cm H (0.24''
OD.times.12''H) pH probe (other designs employed a shorter pH probe
(12 mm.times.152 mm)), a sample/fill port via 0.476 cm
OD.times.0.159 cm ID Tygon tubing ( 3/16'' OD.times. 1/16'' ID), an
organic carbon liquid (e.g., acetic acid) injection via 0.3 cm
OD.times.0.1 cm ID Tygon tubing (0.117'' OD.times.0.039'' ID), a
thermistor 0.0.635 cm OD.times.3.81 cm L @30.48 cm depth (0.25''
OD.times.1.5''L @12'' depth), and a 0.254 cm (0.1'') hole for
venting. The probes were placed at a position to be submerged into
the culture medium contained in the glass culture vessels. A 24V DC
peristaltic pump rated at 1 ml/min flow was used to pump organic
carbon liquid to the columns. A 0-10.OMEGA. potentiometer was used
to control the peristaltic pump speed. The pump was actuated using
a signal from a Hanna pH controller (these controllers have a
hysteresis of 0.1). A 50 ml polypropylene centrifuge tube was used
as the acetic acid reservoir. Aeration was controlled via a
rotameter (0.7 L/min maximum flow). A Luer Lock was placed on the
end of the sample/fill Tygon tubing. Lighting was provided via an 8
bulb T5 fluorescent bulb light fixture. The fluorescent bulbs apply
light from about 50 to 500 microeinsteins/m.sup.2s. The axes of the
light tubes were aligned perpendicular to the vertical axis of the
each culture vessel for this specific unit. Alternatively, the
lights can be aligned parallel to the vertical axis of each culture
vessel. A box fan was placed adjacent to the lights blowing toward
the columns to help remove some of the heat from the lights. Data
logging is accomplished by way of a C-RIO and NI software
[0064] Initially there were no controls for the CO.sub.2; it was
fed at a slow constant rate (app. 0.02 LPM). An optional water bath
type cooling system made of a 2 ft by 2 ft (61 cm by 61 cm) flat
panel made of clear acrylic was used as the bath. A submersible
pump was placed into an insulated reservoir and pumped water
through a 2400 btu/h chiller on its way to the water bath. From the
water bath water flowed back to the reservoir. The water bath
system is able to hold temperatures to 20.degree. C. (no heating
capability).
Additional Bioreactor Array Embodiments
[0065] An alternative system may mount each culture vessel on an
aluminum block with a Peltier temperature control system, which is
independently variable instead of a water bath.
[0066] Another bioreactor array design may comprise multiple
variations. LED lights may be added in place of the fluorescent
lights and use multiple diodes (e.g., red, far red, blue) with
wavelengths that range from about 300 to about 800 nm. Each diode
may be dimmed and variably controlled independent of one another. A
second modification may be a set of overflow ports placed at 600,
700, and 800 ml on each 1000 ml column. An overflow reservoir may
be added and plumbed to the desired overflow ports. The unused
ports may be capped.
[0067] In another bioreactor array embodiment, the water bath
cooling system may be replaced by thermoelectric cooling. The
column stand for this system may be fully enclosed with two clear
plexiglass faces in front and back for viewing and lighting the
columns. The top, bottom, and sides may be constructed from 0.75
inch (2 cm) plywood to help insulate the chamber. The chamber may
be split down the center with 0.75 inch (2 cm) plywood creating two
chambers that house 4 column reactors and 4 acid reservoirs per
chamber. Thermoelectric coolers (app. 80-100 btu/h) may be added to
each chamber and controlled via simple thermostat. LED lights may
be employed in place of the fluorescent lights. These lights may
have multiple diodes (e.g., red, blue, and white diodes) that may
independently controlled as for intensity, photoperiod, and other
parameters. A variable voltage power supply may be used to power
the liquid delivery pumps. This allows the pumps to be sped up or
slowed down easily with no need for resistors. Rotameters may be
added and plumbed into the sparger air to facilitate the use of an
additional gas.
Example 2: Control of the Bioreactor Array
[0068] The bioreactor array system of Example 1 was fitted with a
programmable logic controller (PLC) controls/data logging system.
The PLC is used to control temperature, pH, dissolved oxygen (DO),
light intensity/spectrum, light cycle, and growth mode
(phototrophic, heterotrophic, mixotrophic) all of which can be
manipulated through a user friendly touch screen mounted directly
to the unit. This system uses Peltier devices for heating/cooling,
which can be used for climate control or can be mounted to the
column directly for individual temperature control. Controls for
CO.sub.2 as well as acid or alkali injection for pH control are
used. There are multiple versions of harvest systems that can be
employed on this system (e.g., manual, overflow, pumped) depending
on operator choice. An inert gas can be added to sparger gas to
help control DO via a feed back control loop between a DO probe and
solenoid to control gas flow. A continuous media addition system
can be added for concentration control and are driven by a signal
from an optical density type sensor. Media and acid consumption are
monitored by load sensors installed on the reservoirs.
Example 3: Bioreactor Array Operation
[0069] The column bioreactor array system of Example 1 is used to
determine whether an antimicrobial gas treatment can prevent or
treat cultures that were contaminated. A single Chlorella sp.
strain is added to all vessels along with conventional BG-11 growth
medium at a concentration of 1 g/l. A harmful contaminant
Polyarthra vulgaris, is added to each culture vessel at the
designated time shown in Table 1. Various concentrations of
antimicrobial gases (i.e., ozone, chlorine dioxide, ethylene oxide)
are mixed with carbon dioxide enriched air and bubbled through the
culture vessels. The array of vessels are grown for a 10 day cycle
under 12 hours light/12 hours darkness for 5 days using
nutrient-sufficient medium (growth phase) followed by 5 days
without a nitrogen source in the medium (oil accumulation phase).
The conditions and all other parameters are held constant with a
common light source and common lines delivering the same amounts of
solids, liquids and gases to each. The combinations of culture
treatments are given in Table 1.
TABLE-US-00001 TABLE 1 Culture vessel Time of adding number Gas
treatment Gas concentration contaminant 1 (control) None None
Initial 2 (control) None None 3 days 3 (control) None None 8 days 4
Ozone Low Initial 5 Ozone Low 3 days 6 Ozone Low 8 days 7 Ozone
Intermediate Initial 8 Ozone Intermediate 3 days 9 Ozone
Intermediate 8 days 10 Ozone High Initial 11 Ozone High 3 days 12
Ozone High 8 days 13 Chlorine dioxide Low Initial 14 Chlorine
dioxide Low 3 days 15 Chlorine dioxide Low 8 days 16 Chlorine
dioxide Intermediate Initial 17 Chlorine dioxide Intermediate 3
days 18 Chlorine dioxide Intermediate 8 days 19 Chlorine dioxide
High Initial 20 Chlorine dioxide High 3 days 21 Chlorine dioxide
High 8 days 22 Ethylene oxide Low Initial 23 Ethylene oxide Low 3
days 24 Ethylene oxide Low 8 days 25 Ethylene oxide Intermediate
Initial 26 Ethylene oxide Intermediate 3 days 27 Ethylene oxide
Intermediate 8 days 28 Ethylene oxide High Initial 29 Ethylene
oxide High 3 days 30 Ethylene oxide High 8 days 31 Ozone Low None
32 Ozone Intermediate None 33 Ozone High None 34 Chlorine dioxide
Low None 35 Chlorine dioxide Intermediate None 36 Chlorine dioxide
High None 37 Ethylene oxide Low None 38 Ethylene oxide Intermediate
None 39 Ethylene oxide High None 40 (control) None None None
[0070] The differing gas treatments without adding a contaminant
serve as a control for determining the baseline of inhibitory
effects of the gas on the microalgae. Likewise the differing time
for inoculation with the contaminant serve to determine a baseline
of the harmful effects on the culture depending on the growth
cycle. The differing gasses and their differing concentrations
serve as techniques being optimized between the harmful effects on
the microalgae and the beneficial harmful effects on the
contaminating microbe.
[0071] At the end of the 10 day cycle the resulting culture liquid
is centrifuged and dried, the biomass weighed, the protein content
estimated by the Comassie Blue method and the lipid content
estimated by the Nile Red method (Cooksey et al, (1987)). The
entire process can be completed in two weeks as compared to a year
or more using a single system with the potential for changing
conditions (especially light conditions) and instrument drift
during that time.
[0072] The method may be repeated with any combination of
photosynthetic microorganisms and contaminant and may be repeated
where the antimicrobial gas is not added until after the
contaminant has been added and is detectably affecting the cell
growth. This approach may determine whether any antimicrobial gas
treatment can rescue a contaminated culture. Further methods may be
run with other combinations of microorganisms and varied parameters
to draw other conclusions in a similar manner and short time
frame.
[0073] Previous systems using the 2 ft (61 cm) by 2 ft (61 cm) flat
panel reactors with a volume of 10-15 L required about 15 g of
biomass to inoculate each reactor. Combinatorial experiments using
eight reactors would require about 120 g of biomass and about 120 L
of prepared media. In one embodiment of bioreactor array system,
the culture vessel may have an operating volume of 500 to 1000 ml,
which requires only 0.5-1.5 g, or about 1 g, of biomass to
inoculate each reactor. Combinatorial experiments using eight
culture vessels of the instant invention would require about 8 g of
biomass and about 4-8 L of prepared media. The dramatic reduction
in biomass and culture media resources required allows for more
experiments to be done, but still produces the minimum amount of
biomass required for composition analytical tests to be performed.
The results from the composition analytical tests may be used for
product development and verification of parameters for larger scale
bioreactors.
[0074] The capability for each culturing vessel in the bioreactor
array to be have individual and independently controllable organic
carbon, lighting, and gas supply systems provides the flexibility
to operate in phototrophic, mixotrophic, and heterotrophic
culturing conditions. In some embodiments, the culturing vessel may
receive light and air or carbon dioxide gas, but no organic carbon
to operate in phototrophic culture conditions. In some embodiments,
the culturing vessel may receive light, organic carbon, and gases
(e.g., air, carbon dioxide, oxygen) to operate in mixotrophic
culture conditions. In some embodiments, the culturing vessel may
receive organic carbon and oxygen or air, but no light to operate
in heterotrophic conditions. Parameters may be independently
controlled in the bioreactor array as described through the
specification to produce biomass and combinatorial testing results
in any of the described culture conditions. Dissolved oxygen (DO)
control becomes important in mixotrophic and heterotrophic
conditions due to the consumption of oxygen by the microorganisms
and high cell densities that may result from rapid growth. An inert
gas may be added to the sparger air to help control the dissolved
oxygen via a DO probe and a solenoid to control gas flow.
[0075] Multiple available harvest systems may comprise a manual
harvest system, an overflow harvested system set a desired volume
(e.g., 600 ml, 700 ml, 800 ml), or a pumped harvest system. The
harvest systems may be controlled for different harvesting rates
from the different culture vessels by methods such as, but not
limited to: controlling the harvesting pump devices at different
settings, and setting the overflow volume level at different volume
levels in different culture vessels. Continuous media addition may
be added for cell concentration control independently of the
harvest system or in combination with the harvest system, and may
be driven by a signal from an optical density sensor.
[0076] In one embodiment the bioreactor array may comprise a
plurality of culture vessels configured to contain an aqueous
culture of microorganisms in an interior volume of the culture
vessel. Each culture vessel in the bioreactor array may comprise an
independently controlled supply of gas, nutrients, and light. The
gases may comprise air, oxygen, carbon dioxide, nitrogen, and/or
other inert gases. The supply of gases may be controlled to
maintain levels of dissolved carbon dioxide, dissolved oxygen, pH,
and mixing by aeration. The supply may be controlled as to the type
of gas, volume of gas, concentration of gas, flow rate, and bubble
size. The nutrients may comprise minerals in an aqueous medium
and/or an organic carbon source. The light may be supplied by
lighting devices disposed outside the culture vessel, within the
interior volume of the culture vessel, or combinations thereof. The
independently controlled light supply may be completely turned off
for heterotrophic culture conditions, or varied in amount,
intensity, photoperiod, light/dark cycle, and wavelength of light
for phototrophic and mixotrophic culture conditions. In some
embodiments, the lighting device may comprise at least two light
emitting diodes (LEDs) that provide different wavelengths of light
and are independently controllable.
[0077] In some embodiments, each culture vessel may further
comprise an independently controllable heat exchanger to control
temperature of the culture. In some embodiments, the culture vessel
may be translucent, transparent or clear, which facilitates the use
of a lighting device disposed outside of the culture vessel. In
some embodiments, the culture vessel comprises at least one opaque
section which blocks light exterior to the culture vessel. A
culture vessel with at least one opaque section may reduce light
for mixotrophic conditions, block all light for heterotrophic
conditions, or facilitate the use of a lighting device disposed
within the interior volume of the culture vessel.
[0078] In some embodiments, the bioreactor array may further
comprise an independently controlled harvesting system for each
culture vessel. Multiple available harvest systems may comprise a
manual harvest system, an overflow harvested system set a desired
volume (e.g., 600 ml, 700 ml, 800 ml), or a pumped harvest system.
The harvest systems may be controlled for different harvesting
rates from the different culture vessels by methods such as, but
not limited to: controlling the harvesting pump devices at
different settings, and setting the overflow volume level at
different volume levels in different culture vessels. Continuous
media addition may be added for concentration control independently
of the harvest system or in combination with the harvest system,
and may be driven by a signal from an optical density sensor.
[0079] In some embodiments, the bioreactor array further comprises
at least one sensor configured to measure at least one culturing
parameter of each culture vessel. The at least one sensor may be
selected from the group consisting of: pH sensor, temperature
sensors, light sensors, dissolved oxygen sensors, dissolved carbon
dioxide sensor, and optical density sensor. Sensors at each
culturing vessel may be coordinated for control of the other
features of the bioreactor array through a programmable logic
controller, and for data recording through a data logging
device.
[0080] Embodiments of the bioreactor array may be used in methods
of culturing microorganisms, particularly for combinatorial testing
of different parameters for cultures of the same microorganisms,
the same parameters for cultures of different microorganisms, and
combinations thereof. In one embodiment, a method comprises
providing a culture of microorganisms in an aqueous culture medium
in a plurality of culture vessels; independently controlling the
supply of at least one selected from the group consisting of light,
at least one gas, at least one nutrient, and heat exchange to each
culture vessel; and wherein each culture vessel contains a culture
of the same microorganisms with different parameters. The
parameters may be selected from the group consisting of
temperature, pH, amount of light, intensity of light, wavelengths
of light, light photoperiod, light/dark cycle, concentration of
gases, and agitation from gas supply. For embodiments with
harvesting systems, the harvesting rate may also be an
independently controlled parameter for each culture vessel.
[0081] It will be understood that various modifications may be made
to the embodiments disclosed herein. Therefore, the above
description should not be construed as limiting, but merely as
exemplifications of preferred embodiments. Those skilled in the art
will envision other modifications within the scope and spirit of
the claims appended hereto. All patents and references cited herein
are explicitly incorporated by reference in their entirety.
* * * * *