U.S. patent application number 14/466388 was filed with the patent office on 2015-05-21 for polyhydroxyalkanoate production and related processes.
The applicant listed for this patent is Newlight Technologies, LLC. Invention is credited to Evan Creelman, Markus Herrema, Kenton Kimmel.
Application Number | 20150140621 14/466388 |
Document ID | / |
Family ID | 43732751 |
Filed Date | 2015-05-21 |
United States Patent
Application |
20150140621 |
Kind Code |
A1 |
Herrema; Markus ; et
al. |
May 21, 2015 |
POLYHYDROXYALKANOATE PRODUCTION AND RELATED PROCESSES
Abstract
Embodiments of the invention relate generally to processes for
the production and processing of polyhydroxyalkanoates (PHA) from
carbon sources. In several embodiments, PHAs are produced at high
efficiencies from carbon-containing gases through the utilization
of a regenerative polymerization system.
Inventors: |
Herrema; Markus; (Newport
Beach, CA) ; Kimmel; Kenton; (Dana Point, CA)
; Creelman; Evan; (Costa Mesa, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Newlight Technologies, LLC |
Irvine |
CA |
US |
|
|
Family ID: |
43732751 |
Appl. No.: |
14/466388 |
Filed: |
August 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13392502 |
Feb 24, 2012 |
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PCT/US2010/047052 |
Aug 27, 2010 |
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14466388 |
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61237606 |
Aug 27, 2009 |
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61237609 |
Aug 27, 2009 |
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61237635 |
Aug 27, 2009 |
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61237603 |
Aug 27, 2009 |
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61237616 |
Aug 27, 2009 |
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61237615 |
Aug 27, 2009 |
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61237620 |
Aug 27, 2009 |
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61237643 |
Aug 27, 2009 |
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61237633 |
Aug 27, 2009 |
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61237630 |
Aug 27, 2009 |
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61237626 |
Aug 27, 2009 |
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61237642 |
Aug 27, 2009 |
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61237639 |
Aug 27, 2009 |
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61237627 |
Aug 27, 2009 |
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Current U.S.
Class: |
435/135 |
Current CPC
Class: |
C12P 7/625 20130101;
C12N 1/00 20130101; C08G 63/90 20130101; C08G 63/06 20130101; C08G
63/89 20130101 |
Class at
Publication: |
435/135 |
International
Class: |
C12P 7/62 20060101
C12P007/62 |
Claims
1.-18. (canceled)
19. A process for producing polyhydroxyalkanoate (PHA) by a culture
of microorganisms, the process comprising: (a) providing a culture
of two or more strains of microorganisms comprising a first strain
of methanotrophic microorganisms capable of metabolizing methane to
synthesize PHA and a second strain of microorganisms capable of
metabolizing a PHA-reduced biomass to synthesize PHA; (b) providing
a culture medium comprising at least one nutrient, methane, and a
PHA-reduced biomass; (c) exposing said culture to said culture
medium; (d) subjecting said culture to a growth period causing said
first strain to use said methane to reproduce and said second
strain to use said PHA-reduced biomass to reproduce; and (e)
subjecting said grown culture to a polymerization period by
reducing the concentration of at least one of said nutrients
thereby causing said first strain to use said methane to synthesize
PHA and said second strain to use said PHA-reduced biomass to
synthesize PHA.
20. The process of claim 19, further comprising the step of
removing a portion of said culture following said polymerization
period.
21. The process of claim 20, further comprising the step of
extracting said PHA from said removed culture thereby producing a
PHA extract and a PHA-reduced biomass.
22. The process of claim 19, wherein said methanotrophic
microorganism further metabolizes gases, including carbon dioxide,
volatile organic compounds, air and/or oxygen.
23. The process of claim 21, further comprising the step of
returning said PHA-reduced biomass to said culture medium of claim
19, step (b) to serve as a carbon source for said second strain of
microorganisms.
24. The process of claim 19, wherein said culture of microorganisms
comprise a mixed culture of microorganisms comprising
carbon-dioxide utilizing microorganisms, heterotrophic
microorganisms, autotrophic microorganisms, cyanobacteria,
biomass-utilizing microorganisms, methanogenic microorganisms,
aerobic microorganisms, anaerobic microorganisms, acidogenic
microorganisms, and/or acetogenic microorganisms.
25. The process of claim 23, wherein said PHA-reduced biomass is
metabolized as assimilable sources of carbon and converted into
said PHA.
26. The process of claim 25, wherein said carbon within said
PHA-reduced biomass is metabolized by said second culture to
produce carbon dioxide and/or methane whereby said carbon dioxide
and/or said methane is further metabolized by said first strain of
microorganisms capable of metabolizing methane in the production of
said PHA.
27. The process of claim 21, wherein extracting said PHA from said
removed culture comprises mixing said removed culture with an
extraction agent or mechanism selected from the group consisting of
solvents, solvent washing, chemical treatment, microwave treatment,
simple or fractional distillation, supercritical carbon dioxide,
heat, enzymes, surfactants, acids, bases, hypochlorite, peroxides,
bleaches, ozone, EDTA, and/or a combinations thereof.
28. The process of claim 27, wherein said solvent is selected from
the group consisting of methylene chloride, acetone, ethanol,
methanol, ketones, alcohol, chloroform, dichloroethane, water,
carbon dioxide, and/or a combinations thereof.
29. The process of claim 27, wherein said mechanism is selected
from the group consisting of sonication, homogenization,
distillation, spray drying, hypochlorite non-PHA dissolution,
protonic non-PHA dissolution, non-PHA dissolution, enzymatic
treatment, and/or freeze drying.
30. The process of claim 19, further comprising the step of
introducing a light during said growth period.
31. The process of claim 30, wherein said light is introduced using
light emitting device to influence the metabolism of said
culture.
32. The process of claim 30, wherein said light is emitted by a
device that is charged by inserting the two leads of a 115V AC
power source into said culture medium.
33. The process of claim 19, wherein said methane is derived from
one or more sources from the group consisting of landfills,
wastewater treatment plants, power production facilities or
equipment, agricultural digesters, oil refineries, natural gas
refineries, natural gas streams, cement production facilities,
and/or anaerobic organic waste digesters.
34. A process for polyhydroxyalkanoate (PHA) production by a
culture of microorganisms, the process comprising: (a) fermenting a
culture of at least two strains of microorganisms comprising a
culture medium, a first strain of microorganism capable of
metabolizing a carbon-containing gas, a second strain of
microorganism capable of metabolizing a reduced biomass, and at
least one nutrient; (b) exposing said fermenting culture of
microorganisms to an excess supply of carbon-containing gas
throughout said fermentation step; (c) subjecting said fermenting
culture of microorganisms to a growth period causing said culture
of fermenting microorganisms to reproduce; (d) subjecting said
fermenting culture of microorganisms to a polymerization period by
reducing the concentration of at least one of said nutrients
thereby causing said reproduced culture capable of synthesizing PHA
to synthesize PHA; (e) repeatedly cycling said culture of
microorganisms between said growth period and said polymerization
period until the desired quantity of intracellular PHA is produced
by those microorganisms capable of producing PHA thereby producing
a PHA-containing biomass; (f) removing a portion of said
microorganisms following step (e) to harvest said PHA to create a
reduced biomass; (g) returning said reduced biomass produced in
step (f) to the culture of step (a), wherein reducing the
concentration of said one or more nutrients in step (c) and
supplying an excess quantity of carbon-containing gas further
causes said first and second strain of microorganism to synthesize
PHA from said carbon-containing gas and said reduced biomass
respectively; and (h) repeating steps (a)-(h) at least two times,
to increase the yield of PHA produced using only said carbon
containing gas and said reduced biomass as a carbon source.
35. The method of claim 34, wherein said culturing is performed
under non-sterile conditions.
36. The method of claim 34, wherein said PHA concentrations are
least 80% of total dry cell weight of said first strain of
microorganism.
37. The method of claim 34, wherein said additional nutrient
comprises at least one of the nutrients selected from the group
consisting of aluminum, boron, calcium, carbon, carbon dioxide,
cobalt, iron, magnesium, molybdenum, nitrogen, oxygen, phosphorus,
potassium, sodium, and zinc.
38. The method of claim 34, wherein said first strain of
microorganism capable of metabolizing a carbon-containing gas
comprises methanotrophic microorganisms of a genus selected from a
group consisting of: Methylosinus, Methylocystis, Methylococcus,
Methylobacterium, and Pseudomonas.
39. The method of claim 34, wherein at least one of said additional
nutrient comprises dissolved oxygen and wherein the method further
comprises increasing the concentration of dissolved oxygen in said
culture media to preferentially select for methanotrophic
microorganisms exhibiting reduced pigmentation.
40. The method of claim 34, further comprising purifying said
extracted PHA.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of co-pending U.S. patent
application Ser. No. 13/392,502 filed Feb. 24, 2012, which is the
U.S. National Phase of International Application No.
PCT/US2010/047052, filed Aug. 27, 2010, which claims the benefit of
U.S. Provisional Application Nos. 61/237,606, 61/237,609,
61/237,635, 61/237,603, 61/237,616, 61/237,615, 61/237,620,
61/237,643, 61/237,633, 61/237,630, 61/237,626, 61/237,642,
61/237,639, and 61/237,627, all filed on Aug. 27, 2009, the entire
disclosure of each application in the priority chain is
incorporated by reference herein.
BACKGROUND
[0002] 1. Field of the Invention
[0003] Embodiments of the invention relate to an improved process
for the production and processing of polyhydroxyalkanoates, and
specifically to a process for the production of
polyhydroxyalkanoates from carbon-containing gases.
[0004] 2. Description of the Related Art
[0005] Polyhydroxyalkanoates (PHAs) are thermoplastic polyesters
that serve as energy storage vehicles in microorganisms. PHAs are
biodegradable in both aerobic and anaerobic conditions, are
biocompatible with mammalian tissues, and, as thermoplastics, can
be used as alternatives to fossil fuel-based plastics such as
polypropylene, polyethylene, and polystyrene. In comparison to
petrochemical-based plastics, which are neither biodegradable nor
made from sustainable sources of carbon, PHA plastics afford
significant environmental benefits.
[0006] The utilization of food crop derived sugars in genetically
engineered microorganism-based aqueous fermentation systems is
often regarded as the most efficient and economical platform for
PHA production. Specifically, sugar-based PHA production processes
are capable of generating high density fermentation cultures and
high PHA inclusion concentrations, and, by maximizing the cell
culture density and PHA inclusion concentration therein, it is
believed that carbon, chemical, and energy efficiencies are also
maximized. For example, comparing a low cell and PHA concentration
process to a high cell and PHA concentration process, a low
concentration process requires significantly more, per given unit
of PHA-containing biomass, i) energy for dewatering cells prior to
PHA extraction treatment, ii) liquid culture volume, and associated
chemicals, mixing energy, and heat removal energy, and iii) both
energy and chemicals for separating PHA from biomass. Accordingly,
whereas the sugar-based genetically-engineered microorganism PHA
process yields maximized cell densities and PHA concentrations
relative to low concentration processes, it is also regarded as the
most carbon, chemical, energy, and, thus, cost efficient PHA
production method.
[0007] Unfortunately, despite these maximized efficiency
advantages, sugar-based PHA production remains many times more
expensive than fossil fuel-based plastics production. Thus, given
the apparent efficiency maximization of the high density
sugar-derived PHA production process, PHAs are widely considered to
be fundamentally unable to compete with fossil fuel-based plastics
on energy, chemical, and cost efficiency.
SUMMARY
[0008] Despite the environmental advantages of PHAs, the high cost
of PHA production relative to the low cost of fossil fuel-based
plastics production has significantly limited the industrial
production and commercial adoption of PHAs.
[0009] To reduce the carbon input cost of the PHA production
process, carbon-containing industrial off-gases, such as carbon
dioxide, methane, and volatile organic compounds, have been
proposed as an alternative to food crop-based sources of carbon. In
addition to the wide availability and low cost of carbon-containing
gases, carbon-containing gases also do not present the
environmental challenges associated with food crop-derived sources
of carbon. Specifically, whereas food crop-based carbon substrates
require land, fertilizers, pesticides, and fossil fuels to produce,
and also generate greenhouse gas emissions during the course of
production, carbon-containing off-gases do not require new inputs
of land, fertilizers, pesticides, or fossil fuels to generate.
Thus, on both an economic and environmental basis, the utilization
of carbon emissions for the production of PHA would appear to offer
significant advantages over sugar-based PHA production
processes.
[0010] Unfortunately, the fermentation of carbon-containing gases
presents technical challenges and stoichiometric limitations that
have, in the past, rendered the gas-to-PHA production process
significantly more energy and chemical intensive, and thus more
costly, than the food crop-based PHA production process.
Specifically, these technical challenges and stoichiometric
limitations include: low mass transfer rates, low microorganism
growth rates, extended polymerization times, low cell densities,
high oxygen demand, and low PHA cellular inclusion concentrations.
Whereas sugar-based fermentation systems have the ability to
generate high cellular densities and PHA inclusion concentrations,
based on cell morphology and mass transfer constraints,
carbon-containing gas-based fermentation processes typically
generate 10-30% of the biomass and intracellular PHA inclusion
concentrations achieved in sugar-based processes. As a result, the
ratio of energy-to-PHA required to carry out upstream carbon
injection, optional oxygen injection, and culture mixing, as well
as downstream PHA purification, significantly exceeds the
energy-to-PHA ratio required for sugar-based PHA production
methods, thereby rendering the emissions-based process
uncompetitive when compared to both petroleum-based plastics and
sugar-based PHAs.
[0011] In light of the potential environmental advantages and
carbon cost efficiencies of utilizing carbon-containing gases as a
source of carbon for PHA production, there exists a significant
need to reduce the energy, chemical, and carbon input-to-PHA output
ratio in a carbon emissions-based PHA production system, and
thereby render carbon gas-derived PHA economically competitive with
petrochemical-based plastics.
[0012] Thus, in several embodiments, the present invention relates
to a novel process for the conversion of carbon-containing gases
into PHAs at previously unattainable energy and carbon PHA
conversion ratios.
[0013] In some embodiments, the invention also relates to a process
that generates a carbon emissions-based PHA material that is
cost-competitive with both food crop-based PHAs and fossil
fuel-based thermoplastics.
[0014] While PHAs are widely considered to be noncompetitive with
fossil fuel-based plastics on energy, chemical, and cost
efficiency, several embodiments of the invention relate to a
process for producing PHAs from carbon-containing gases that yields
unexpectedly improved energy, carbon, chemical, and cost
efficiencies over sugar-based PHA production methodologies.
[0015] More specifically, certain embodiments of the invention
provide high efficiency, high density, high PHA concentration
processes for the production of PHA from carbon-containing gases,
comprising the steps of: (a) providing a microorganism culture
comprising PHA-containing biomass, (b) removing a portion of the
PHA-containing biomass from the culture, (c) extracting a portion
of PHA from the removed culture to produce isolated PHA and
PHA-reduced biomass, (d) purifying the isolated PHA, and (e)
returning the PHA-reduced biomass to the culture to cause the
culture to convert the carbon within the PHA-reduced biomass into
PHA. In several embodiments, carbon output from the system is
wholly or substantially only in the form of PHA.
[0016] In several embodiments, a system for using a microorganism
culture to convert a carbon-containing gas into PHA at high
efficiencies is provided. Microorganisms are cultured using a
combination of one or more carbon-containing gases and PHA-reduced
biomass, or derivatives thereof, as sources of carbon to produce
PHA-containing biomass. A portion of the PHA-containing biomass is
then removed from the culture, and PHA is extracted from the
removed PHA-containing biomass to create substantially PHA-reduced
biomass and substantially isolated PHA.
[0017] Typically, PHA is present in the PHA-containing biomass of
gas-utilizing microorganisms at concentrations in the range of
about 5%-60%, and approximately 40-95% of the PHA-containing
biomass is discarded from the system following PHA extraction. In
some cases, PHA is present in gas-utilization microorganisms in the
range of about 1-90%, including at about 1%, 3%, 5%, 7%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%, and
approximately 10-99% of the PHA-containing biomass is discarded
from the system following PHA extraction, including 99%, 97%, 95%,
93%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30%, 20%, or
10% of the PHA-containing biomass. Rather than discarding the
remaining, e.g., 40-95% of the PHA-reduced biomass, in one
embodiment of the invention, the PHA-reduced biomass is returned
back to the microorganism culture to be regenerated as PHA by a
microorganism culture capable of utilizing PHA-reduced biomass, or
a derivative thereof, as a source of carbon for PHA production,
thereby creating a regenerative closed-loop polymerization system.
By using PHA-reduced biomass as a source of carbon for PHA
production in microorganisms growing as or in association with
gas-utilizing microorganisms, PHA can be produced from
carbon-containing gases at surprisingly and unexpectedly improved
carbon, energy, and chemical efficiencies, since carbon from
carbon-containing gases that would otherwise be discarded is
regenerated as PHA in a microorganism culture, and microorganisms
that produce PHA from carbon-containing gases at low concentrations
(e.g., 5-60% PHA by weight, or less than 70% PHA by weight) can, in
some embodiments, be utilized to produce PHA at significantly
increased carbon-to-PHA efficiencies. In some embodiments, the
regeneration step is repeated to form an essentially closed-loop
system. Thus, in some embodiments, the carbon output from the
system is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 95%, or 99% PHA In other words, at least 1%, 5%, 10%, 20%,
30%, 40%, 50%, 70%, 80%, 90%, 95%, or 99% of the carbon entering
the system is converted into PHA. In other embodiments, 1-5%,
5-10%, 10-20%, 20-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%,
80%-90%, 90%-95%, 95%-99% (and overlapping ranges thereof) of the
carbon entering the system is converted to PHA. By regenerating
PHA-reduced biomass as PHA in a microorganism culture, the
percentage of carbon from a carbon containing gas that is converted
to PHA is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,
8-fold, 9-fold, 10-fold, or greater than systems that do not employ
the regenerative or closed-loop system disclosed herein. In some
embodiments, the regeneration (e.g. return and/or recycling of the
PHA-reduced biomass) step is repeated at least 2, 3, 4, 5, 6, 7, 8,
9, or 10 (or more) times. In some embodiments, the regeneration
step is repeated until at least 90% to 95% of the carbon input into
the system is converted into PHA. In some embodiments, the
regeneration step is repeated as many times as desired to reach a
particular percentage conversion of carbon to PHA.
[0018] Several embodiments of the invention provide for the
production of PHA from carbon-containing gases at previously
unattainable energy, carbon, and chemical efficiencies by way of
providing a microorganism culture capable of metabolizing the
carbon within both a carbon-containing gas and PHA-reduced biomass,
manipulating the conditions of the culture to cause the culture to
produce PHA, removing a portion of PHA-containing biomass from the
culture, extracting the PHA within the removed PHA-containing
biomass to create substantially isolated PHA and substantially
PHA-reduced biomass, returning the PHA-reduced biomass to the
culture and contacting the PHA-reduced biomass with the culture to
cause the culture to metabolize the carbon within the PHA-reduced
biomass into PHA, and purifying the isolated PHA. Thus, an
advantage of several embodiments of the invention is the production
of PHA from carbon-containing gases at significantly improved
energy, carbon, and chemical efficiencies.
[0019] The process according to several embodiments disclosed
herein yields a range of surprising benefits over current gas-based
PHA production technologies. To begin, whereas the cell density of
gas-based fermentation processes is traditionally limited by the
mass transfer or diffusion rates of one or more factors, such as
light, oxygen, carbon dioxide, methane, or volatile organic
compounds, several embodiments disclosed herein enable the
generation of cell densities that significantly exceed cell
densities attainable in the current practice (e.g., by more than
1%, 10%, 20%, 30%, 50%, 80%, 100% or more), and thereby enables
cost-efficient system mixing, aeration, heat control, and
dewatering. For example, current methane-based PHA production
systems are known to be capable (based on cell morphology and mass
transfer characteristics) of generating approximately 60 g/L of
biomass with an overall PHA concentration of 55%, or 33 g/L PHA. In
contrast, in several embodiments of the invention, cell densities
of approximately 135 g/L with an overall PHA concentration of 70%,
or 94.5 g/L PHA are generated in a methane-based PHA production
system. In some embodiments, cell densities of approximately 10
g/L, 20 g/L, 30 g/L, 60 g/L, 75 g/L, 100 g/L, 125 g/L, 135 g/L, 150
g/L or greater are achieved. In some embodiments, overall PHA
concentration in such cultures ranges from approximately 1% to 20%,
20% to 30%, 30% to 55%, 55% to 60%, 65% to 70%, 70% to 80%, and
overlapping ranges thereof result. In several embodiments, such PHA
concentration ranges represent significant, unexpected, and
surprising improvements over traditional processes, e.g., processes
that are limited to low cell densities and/or PHA
concentrations.
[0020] As an non-limiting example of the impact of this improvement
on energy efficiency, the energy required, on an energy
input-to-PHA output basis, to aerate, mix, and dewater a 135 g/L
solution with a PHA concentration of 70% by weight is 186% less
than the energy required to aerate, mix, and dewater a 60 g/L
microorganism solution comprising 40% PHA by weight. It shall be
appreciated that variations in the energy efficiency gains based on
the systems and processes disclosed herein may occur, depending on
the culture conditions, the strain or organisms used, and the
initial gas stream or other carbon source. In several embodiments,
even modest increases in efficiency have substantial benefits. For
example, the ability to efficiently use an input gas having a low
carbon concentration that would not otherwise be useful in PHA
production may prevent the release of such a gas into the
environment and/or reaction of the gas with other atmospheric
compounds, thereby reducing the adverse impact of the low carbon
concentration gas on the environment (e.g., destruction of ozone,
greenhouse gas emission, pollution, etc.).
[0021] Additionally, whereas current gas-based PHA production
systems produce significant carbon losses as a result of the low
PHA inclusion concentrations of gas-utilizing microorganisms (i.e.,
a significant portion of carbon and energy input is lost as
biomass), several embodiments of the invention enable the
generation of overall carbon input yield efficiencies approaching
maximum substrate values; e.g., 100% carbon input-to-PHA yield,
minus respiration and/or downstream processing losses. In some
embodiments, at least 5%, at least 10%, at least 30%, at least 50%,
at least 70%, or at least 90%, carbon input-to-PHA yield is
achieved. It is one important advantage of several embodiments of
the invention that maximum carbon yield efficiencies are
unexpectedly and surprisingly generated in a PHA production system
employing gas-utilizing microorganisms, and particularly, in some
embodiments, in PHA production systems employing gas-utilizing
microorganisms that produce low biomass and/or PHA inclusion
densities.
[0022] In some embodiments, the microorganism culture is a mixed
culture, comprising heterotrophic microorganisms, methanotrophic
microorganisms, autotrophic microorganisms, bacteria, yeast, fungi,
algae, or combinations thereof. In other embodiments, the
microorganism culture may be one or more cultures (e.g., a
plurality of cultures). In some embodiments, the cultures are grown
in one or more bioreactors. In some embodiments, the bioreactors
utilize one or more culture conditions, including both aerobic and
anaerobic conditions. In some embodiments, the microorganism
culture converts PHA-reduced biomass to methane in an anaerobic
process and subsequently to PHA in an aerobic process, such that
PHA-reduced biomass is first anaerobically metabolized to methane
and then used as methane to produce biomass and PHA in a
methanotrophic culture.
[0023] In several embodiments, at least part of the microorganism
culture is a mixed culture capable of metabolizing
carbon-containing gases, including methane, carbon dioxide,
greenhouse gases, and/or various other volatile organic compounds,
into biomass and/or PHA. In some embodiments, the microorganism
culture comprises a two phase system of anaerobic and anaerobic
metabolism, whereby carbon-containing gas is produced in a first
substantially anaerobic phase and subsequently converted into PHA
in a second phase, wherein the microorganism culture in the first
phase is substantially anaerobic and the culture in the second
phase is either anaerobic or aerobic, wherein the two phases may be
operated in one single vessel or in multiple vessels.
[0024] In some embodiments, at least one or more of the
microorganisms are contacted with artificial and/or natural light
during one or more steps of the methods disclosed herein.
[0025] In some embodiments, at least one of more of the
microorganisms is contacted with dissolved oxygen during one or
more steps of the methods disclosed herein.
[0026] In some embodiments, at least one of more of the
microorganisms is cultured at atmospheric, sub-atmospheric, or
above-atmospheric pressures.
[0027] In some embodiments, at least one of more of the
microorganisms can utilize only a carbon-containing gas as a source
of carbon.
[0028] In several embodiments, at least one of more of the
microorganisms can utilize carbon derived from a PHA-reduced
biomass as a source of carbon. In other embodiments, at least one
or more of the microorganisms is a heterotrophic microorganism
capable of converting PHA-reduced biomass into, carbon dioxide,
oxygen, biomass, and/or PHA.
[0029] In several embodiments, at least one or more of the
microorganisms are cultured using carbon derived from both a
carbon-containing gas and a PHA-reduced biomass.
[0030] In some embodiments, the microorganism culture is a pure
culture. In some embodiments, the cultures are maintained in
semi-sterile or sterile conditions.
[0031] In some embodiments, the microorganism culture is a mixed,
non-sterile culture, including a naturally equilibrating consortium
of microorganisms.
[0032] In several embodiments, the microorganism culture is at
least partially comprised of genetically engineered
microorganisms.
[0033] In some embodiments, the microorganism culture is a mixed
culture comprising a combination of naturally occurring and
genetically engineered microorganisms.
[0034] In several embodiments, the PHA is removed from the
microorganism culture by solvent extraction, including solvent
extraction at temperatures ranging from 0.degree. C. to 200.degree.
C. and at pressures ranging from -30 psi to 200 psi.
[0035] In several embodiments, the PHA is removed from the
microorganism culture through the utilization of ketones, alcohols,
and/or chlorinated solvents.
[0036] In several embodiments, the PHA is removed from the
microorganism culture by hypochlorite digestion and/or
chlorine-based solvent extraction.
[0037] In several embodiments, the PHA is removed from the
microorganism culture by supercritical carbon dioxide
extraction.
[0038] In several embodiments, the PHA is removed from the
microorganism culture by protonic non-PHA cell material
dissolution.
[0039] In several embodiments, the PHA is partially removed from
the microorganism culture to create a PHA-rich phase and a PHA-poor
phase.
[0040] In several embodiments, the PHA is removed from the
microorganism culture to render the PHA substantially free of
non-PHA material, including substantially 5%, 10%, 20%, 30%-40%,
40%-50%, 50%-60%, 60%-70%, 70%-80%, 80-90%, 90-99% or more pure PHA
by weight.
[0041] In several embodiments, the PHA is removed from the
microorganism culture by manipulating the pH of the microorganism
culture.
[0042] In certain embodiments, at least one of more of the
microorganisms are contacted with methane, carbon dioxide, oxygen,
and/or a combination thereof.
[0043] In several embodiments, multiple culture vessels are
employed, such that microorganism growth, PHA synthesis,
PHA-reduced biomass metabolism, and PHA removal are carried out in
separate vessels.
[0044] In other embodiments, microorganism growth, PHA-reduced
biomass metabolism, and PHA synthesis occurs in a single
vessel.
[0045] In still other embodiments, microorganism growth and PHA
synthesis occur in a single vessel and PHA extraction is carried
out in one or more separate vessels.
[0046] In several embodiments of the process as disclosed herein,
PHA synthesis is regulated by manipulating the concentration of a
material in the process, wherein the material is oxygen, methane,
carbon dioxide, nitrogen, phosphorus, copper, iron, manganese,
carbon, magnesium, potassium, cobalt, aluminum, sulfate, chlorine,
boron, citric acid, or EDTA.
[0047] In several embodiments, the microorganism culture comprises
one or more strains of microorganisms collectively capable of
converting the carbon within a carbon-containing gas into cellular
biomass and the carbon from cellular biomass or methane into
PHA.
[0048] In several embodiments, the microorganisms are subjected to
filtration, centrifugation, settling, and/or density
separation.
[0049] In several embodiments, the isolated PHA and/or the
PHA-reduced biomass is subjected to filtration, centrifugation,
settling, and/or density separation.
[0050] In some embodiments, the process further comprises washing
the recovered PHA with water, solvent, or other liquid-based agents
to purify the PHA.
[0051] In several embodiments, the process further comprises
oxidizing the recovered PHA to purify the PHA.
[0052] In several embodiments, the process further comprises drying
the recovered PHA to remove volatiles such as water and/or one or
more solvents.
[0053] In several embodiments of the invention, methods for the
production of PHA are provided. In one embodiment, the method
comprises: (a) providing a microorganism culture comprising
PHA-containing biomass, (b) removing a portion of the
PHA-containing biomass from the culture, (c) extracting a portion
of the PHA from the removed PHA-containing biomass to produce
isolated PHA and PHA-reduced biomass, (d) returning the PHA-reduced
biomass to the culture to cause the culture to convert the carbon
within the PHA-reduced biomass into PHA, and (e) purifying the
isolated PHA.
[0054] In one embodiment, the microorganism culture utilizes the
PHA-reduced biomass, or derivatives thereof, such as carbon
dioxide, methane, or volatile organic acids, volatile fatty acids,
volatile organic compounds, non-methane organic compounds, and one
or more carbon-containing gas as a source of carbon. In one
embodiment, the gas is selected from the group consisting of
methane, carbon dioxide, volatile organic compounds, and
hydrocarbons. In one embodiment, the gas is derived from one or
more sources from the group consisting of: landfills, wastewater
treatment plants, power production facilities or equipment,
agricultural digesters, oil refineries, natural gas refineries,
cement production facilities, and/or anaerobic organic waste
digesters.
[0055] In some embodiments, the carbon in the PHA-reduced biomass
is derived from one or more gases from the group consisting of:
methane, biogas, carbon dioxide, volatile organic compounds,
natural gas, wastewater treatment methane and VOCs, and
hydrocarbons.
[0056] In some embodiments, natural and/or artificial light is
utilized to induce the metabolism of the carbon dioxide by the
culture.
[0057] In some embodiments, the microorganism culture comprises one
strain, or a consortium of strains, of microorganisms, including
one or more microorganisms selected from the group consisting of:
bacteria, fungi, yeast, and algae, and combinations thereof.
[0058] In some embodiments, the microorganism culture comprises one
or more microorganisms from the group consisting of: methanotrophic
microorganisms, carbon-dioxide utilizing microorganisms, anaerobic
microorganisms, methanogenic microorganisms, acidogenic
microorganisms, acetogenic microorganisms, heterotrophic
microorganisms, autotrophic microorganisms, cyanobacteria, and
biomass-utilizing microorganisms, and combinations thereof.
[0059] In some embodiments, at least a portion of the microorganism
culture is naturally occurring. In some embodiments, at least a
portion of the microorganism culture is and/or genetically
engineered. In some embodiments, naturally occurring and
genetically engineered microorganisms are both used in the
culture.
[0060] In some embodiments, the microorganism culture is at least
partially maintained under above-atmospheric pressure.
[0061] In some embodiments, the PHA-containing biomass includes one
or more microorganism-derived materials selected from the group
consisting of: intracellular, cellular, and/or extracellular
material, including a polymer, amino acid, nucleic acid,
carbohydrate, lipid, sugar, polyhydroxyalkanoate, chemical, and/or
metabolic derivative, intermediary, and/or end-product. In some
embodiments, the PHA-containing biomass includes one or more
microorganism-derived materials selected from the group consisting
of: methane, volatile organic compounds, carbon dioxide, and
organic acids.
[0062] In one embodiment, the PHA-containing biomass contains less
than about 95% water, including less than about 90%, 85%, 80%, 75%,
or 70% water.
[0063] In some embodiments, the PHA-containing biomass is mixed
with a chemical, including one or more chemicals from the group
consisting of: methylene chloride, acetone, ethanol, methanol,
ketones, alcohols, chloroform, and dichloroethane, or combinations
thereof.
[0064] In one embodiment, the PHA-containing biomass is processed
through homogenization, heat treatment, pH treatment, enzyme
treatment, solvent treatment, spray drying, freeze drying,
sonication, and microwave treatment, or combinations thereof.
[0065] In one embodiment, the PHA-reduced biomass includes the
PHA-containing biomass wherein at least a portion of the PHA has
been removed from the PHA-containing biomass. In another
embodiment, the PHA-reduced biomass includes methane, carbon
dioxide, and organic compounds produced from the PHA-reduced
biomass.
[0066] In some embodiments, the PHA-reduced biomass is subject to
dewatering, chemical treatment, sonication, additional PHA
extraction, homogenization, sonication, heat treatment, pH
treatment, hypochlorite treatment, microwave treatment,
microbiological treatment, including both aerobic and anaerobic
digestion, solvent treatment, water washing, solvent washing,
and/or drying, including simple or fractional distillation, spray
drying, freeze drying, and/or oven drying, or combinations
thereof.
[0067] In several embodiments, the microorganism culture is
maintained in a sterile, semi-sterile, or non-sterile
environment.
[0068] In one embodiment, the PHA includes one or more PHA selected
from the group consisting of: polyhydroxybutyrate (PHB),
polyhydroxyvalerate (PHV), polyhydroxybutyrate-covalerate (PHB/V),
polyhydroxyhexanoate (PHHx), and short chain length (SCL), medium
chain length (MCL), and long chain length (LCL) PHAs.
[0069] In several embodiments, the metabolism, growth,
reproduction, and/or PHA synthesis of the culture is controlled,
manipulated, and/or affected by a growth medium. In some
embodiments, the bioavailable and/or total concentration of
nutrients within the growth medium, such as copper, iron, oxygen,
methane, carbon dioxide, nitrogen, magnesium, potassium, calcium,
phosphorus, EDTA, calcium, sodium, boron, zinc, aluminum, nickel,
sulfur, manganese, chlorine, chromium, molybdenum, and/or
combinations thereof are manipulated (e.g., increased, decreased,
or maintained) in order to control the metabolism, growth,
reproduction, and/or PHA synthesis of the culture In some
embodiments, a single nutrient in the growth medium is manipulated,
while in some embodiments, more than one nutrient in the growth
medium is manipulated to achieve the desired effect on the
culture.
[0070] In one embodiment, the conversion of the PHA-reduced biomass
into the PHA is induced and/or controlled by manipulating the
composition of the medium. As discussed herein, the conversion of
PHA-reduced biomass into the PHA can be controlled in a
time-dependent manner to maximize the efficiency of conversion. In
some embodiments, conversion to PHA production is induced about
1-12 hours, about 5-15 hours, or about 8-24 hours after PHA-reduced
biomass is re-introduced into the culture. In some embodiments,
longer times, e.g., about 24 hours to several days or weeks, are
employed.
[0071] In one embodiment, the conversion of the PHA-reduced biomass
into the PHA is effected by manipulating the concentration one or
more elements selected from the group consisting of: nitrogen,
methane, carbon dioxide, phosphorus, oxygen, magnesium, potassium,
iron, copper, sulfate, manganese, calcium, chlorine, boron, zinc,
aluminum, nickel, and/or sodium, and combinations thereof.
[0072] In some embodiments, the PHA is at least partially removed
from the PHA-containing biomass using one or more extraction agents
selected from the group consisting of: solvents, including
methylene chloride, acetone, ethanol, methanol, or dichloroethane,
supercritical carbon dioxide, sonication, homogenization, water,
heat, distillation, spray drying, freeze drying, enzymes,
surfactants, acids, bases, hypochlorite, peroxides, bleaches,
ozone, EDTA, and/or combinations thereof.
[0073] In one embodiment, the extraction process is substantially
carried out at intracellular temperatures less than 100.degree. C.
In other embodiments, temperatures for extraction range from about
10.degree. C. to 30.degree. C., from about 30.degree. C. to
50.degree. C., from about 50.degree. C. to 70.degree. C., from
about 70.degree. C. to 90.degree. C., from about 90.degree. C. to
about 120.degree. C., or higher. In another embodiment, cells are
reused for polymerization following the extraction process as
viable cells.
[0074] In one embodiment, the removal of the PHA from the culture
causes the culture to be temporarily deactivated, such that the
culture, or elements thereof, may be further used for the synthesis
of PHA. In certain embodiments, deactivation is beneficial because
it allows for the delay of PHA production, transfer of material to
another production area, and the like. In some embodiments,
deactivation allows a tailored PHA production time frame. In some
embodiments, the reuse of cells for polymerization is beneficial
because it avoids or reduces the need to produce new biomass prior
to polymerization, thereby reducing the carbon, chemical, and
energy requirement of PHA production.
[0075] In one embodiment, a PHA produced according to the several
embodiments described herein is provided.
[0076] In several embodiments, processes for the production of PHA
from a carbon-containing gas are provided. In one embodiment, the
process comprises the steps of: a) providing a growth medium
comprising a microorganism culture capable of utilizing the carbon
within one or more carbon-containing gas and PHA-reduced biomass,
b) manipulating the medium to cause the culture to produce PHA, c)
removing at least a portion of the PHA within the culture to create
substantially isolated PHA and substantially PHA-reduced biomass,
d) purifying the isolated PHA, and e) returning the PHA-reduced
biomass to the culture to cause the culture to metabolize the
carbon within the PHA-reduced biomass into PHA.
[0077] In one embodiment, the carbon-containing gas is selected
from the group consisting of: methane, carbon dioxide, toluene,
xylene, butane, ethane, methylene chloride, acetone, ethanol,
propane, methanol, vinyl chloride, volatile organic compounds,
hydrocarbons, and combinations thereof.
[0078] In one embodiment, the invention comprises a PHA comprising
carbon derived from PHA-reduced biomass, wherein the PHA-reduced
biomass comprises carbon derived from one or more carbon-containing
gas.
[0079] In one embodiment, a PHA derived from a carbon-containing
greenhouse gas, including methane, carbon dioxide, or combinations
thereof, is provided. In some embodiments, use of such a gas is
particularly advantageous, as it allows for the simultaneous
production of PHA at lower energy costs and higher efficiencies,
but also removes a portion of a destructive gas from the
atmosphere. In some embodiments, processes and systems as disclosed
herein are particularly well suited for use near sources of such
gases (e.g., landfills, power production plants, anaerobic
digesters, etc.) for onsite conversion of harmful gasses to a
commercially valuable product.
[0080] In several embodiments, processes for the oxidation of
methane are provided. In one embodiment, the process comprises:
providing a culture of methanotrophic and autotrophic
microorganisms, providing a growth culture medium comprising
dissolved methane and carbon dioxide, and contacting the culture
with light to cause the culture to convert the carbon dioxide into
oxygen, whereby the culture utilizes the oxygen to oxidize the
methane, thereby reducing or eliminating the need for an extraneous
source of oxygen to drive methanotrophic metabolism.
[0081] In some embodiments, the light used to contact the culture
is artificial light. In some embodiments, the light used to contact
the culture is natural light. In other embodiments, combinations of
natural and artificial light are used. In some such embodiments,
wavelengths of artificial light are specifically filtered out or
controlled such that the culture is exposed to a broader or more
controlled overall spectrum of light (e.g., the sum of wavelengths
of natural light and artificial light). In some embodiments, the
source of light also functions to generate heat, which can be used
to maintain optimal culture temperatures. In other embodiments,
light input is regulated by time, such that specific cultures of
autotrophic and/or heterotrophic microorganisms are selected for or
optimized according to the duration and/or pattern of light
injection (e.g., 0-12 or 12-24 hours light injection, 0-12 or 12-24
hours dark incubation, multi-second pulsation, etc.).
[0082] In one embodiment, the addition of light reduces the need
for exogenous oxygen sources. While such embodiments provide an
advantage in reducing costs of input materials, in some
embodiments, an exogenous source of oxygen, including air, is added
to the culture.
[0083] In some embodiments the addition of autotrophic
microorganisms to the culture impacts the metabolism of the
culture. In such an embodiment, the timed and planned addition,
activation, or metabolic enhancement of autotrophic organisms can
be based on the desire for changing the rate of methane
oxidation.
[0084] In some embodiments, a system is used for PHA production
comprising providing i) a culture of autotrophic, methanotrophic,
methanogenic, and/or heterotrophic microorganisms and ii) a first
gas comprising carbon dioxide, methane, volatile organic compounds,
oxygen, and/or other gas, whereby the culture of microorganisms are
caused to used the first gas to generate a second gas comprising
carbon dioxide, methane, oxygen, volatile organic compounds, and/or
other gas, whereby the culture subsequently is caused to utilize
the second gas for the generation of PHA, which can then be
isolated and purified according to several embodiments disclosed
herein. In some embodiments, the first gas can be methane, carbon
dioxide, oxygen, or volatile organic compounds. In other
embodiments, the second gas can be oxygen, methane, carbon dioxide,
or other volatile organic compounds. In some embodiments,
microorganisms can be used to convert carbon dioxide to biomass
which can in turn be used to produce methane, which can be
subsequently used to produce PHA. In other embodiments,
microorganisms can be used to convert carbon dioxide to oxygen
which can in turn be used to produce PHA. As disclosed herein, the
products generated at each of these steps may be recycled (e.g.,
splitting a portion of the autotrophic culture and recycling it to
generate additional biomass, generating reduced-PHA biomass and
recycling it into the methanotrophic culture to generate additional
biomass and additional PHA).
[0085] In several embodiments, processes for producing autotrophic
microorganisms using only methane as a carbon input are provided.
In one embodiment, the process comprises: adding methane, oxygen,
and methane-utilizing microorganisms to a culture of autotrophic
microorganisms, whereby the methane-utilizing microorganisms
convert the methane into carbon dioxide, and whereby the
autotrophic microorganisms utilize the carbon dioxide as a source
of carbon, thereby reducing or eliminating the need for an
extraneous source of carbon dioxide to drive autotrophic
metabolism. In some embodiments, addition of methanotrophic and/or
heterotrophic microorganisms to the culture impacts the metabolism
of the autotrophic microorganisms. As discussed herein, the
purposeful addition of such microorganisms at particular times
allows for specific levels of control over the overall output and
operation of the system.
[0086] In several embodiments, processes for oxidizing methane at
low concentrations are provided. In one embodiment, the process
comprises: culturing methanotrophic microorganisms in a medium
comprising water, dissolved methane, dissolved oxygen, and mineral
salts, adding methanol to the medium at a rate and volume
sufficient to cause the microorganisms to reduce the concentration
of the methane in the medium, whereby substantially all of the
methane within the medium is utilized, thereby enabling
methanotrophic microorganisms to metabolize methane present at low
bioavailable concentrations. In some embodiments, gas containing
less than 20% methane by volume is contacted with the medium. In
some embodiments, gas containing less than 1% methane by volume is
contacted with the medium. In some embodiments, the methanol is
produced by microorganism metabolism.
[0087] In several embodiments, processes for separating water from
microorganism biomass are provided. In one embodiment, the process
comprises: providing biomass mixed with water in a liquid medium,
mixing the medium with a liquid agent selected from the group
consisting of ketones, alcohols, chlorinated solvents, derivatives
thereof, or combinations thereof, and subjecting the mixture to a
filtration step. In several embodiments, this enables the efficient
separation of biomass from water. In some embodiments, such methods
reduce or eliminate the need for centrifugation in the separation
process. In some embodiments, the liquid agent is acetone, ethanol,
isopropanol, and/or methanol. In other embodiments, other liquid
agents that are miscible with water are used to separate the
biomass from the aqueous portion of the mixture. In several
embodiments separation is achieved by centrifugation (high-speed,
low-speed), gravity separation, multi-stage filtration, or
combinations thereof.
[0088] In several embodiments, processes for extracting a
polyhydroxyalkanoate from a PHA-containing biomass are provided. In
one embodiment, the process comprises the steps of: (a) providing a
PHA-containing biomass comprising PHA and water, (b) mixing said
biomass with a solvent at a temperature sufficient to dissolve at
least a portion of said PHA into said solvent and at a pressure
sufficient to enable substantially all or part of said solvent to
remain in liquid phase, thereby producing a PHA-lean biomass phase
and a PHA-rich solvent phase comprising water, PHA and solvent (c)
separating said PHA-rich solvent phase from said PHA-lean biomass
phase at a temperature and pressure sufficient to enable
substantially all or part of said solvent to remain in liquid phase
and prevent substantially all or part of said PHA within said
PHA-rich solvent phase from precipitating into said water, (d)
reducing the pressure or increasing the temperature of said
PHA-rich solvent phase to cause said PHA-rich solvent to vaporize
and said PHA to precipitate or otherwise become a solid PHA
material while maintaining the temperature and/or pressure of the
PHA-rich solvent phase to prevent all or part of the
temperature-dependent precipitation of said PHA into said water,
and (e) collecting said solid PHA material, including optionally
separating said solid PHA material from said solvent and/or said
water.
[0089] In some embodiments, suitable solvents include acetone,
ethanol, methanol, dichloroethane, and/or methylene chloride.
Depending on the solvent selected, in some embodiments, separating
the solid PHA material from solvent and/or water is achieved by
increasing the temperature of the mixture. In other embodiments,
separation is achieved through reducing the pressure of the
solvent, PHA, and/or water. In some embodiments, combinations of
temperature changes and pressure changes are used to optimally
separate solid PHA material from solvent and/or water. In some
embodiments, evaporation of solvent and/or water occurs in a rapid
fashion, thereby reducing the need for temperature or pressure
changes. Advantageously, certain embodiments of the processes
disclosed herein may optionally be carried out in a batch,
semi-continuous, or continuous manner. Thus, the process can be
tailored to the needs of the producer at any given time.
[0090] In several embodiments, processes for modifying the
functional characteristics of a PHA are provided. In one
embodiment, the process comprises providing a first PHA and a
second PHA, wherein the molecular weight of said second PHA is
greater than the molecular weight of said first PHA, and combining
said first PHA with said second PHA to modify the functional
characteristics of both said first PHA and second PHA. In some
embodiments, both the first PHA and the second PHA are PHB, and in
some embodiments, one or more of the first and second PHA comprises
PHB/V. In one embodiment the first PHA and the second PHA is PHB or
PHBV.
[0091] In some embodiments, the molecular weight of the first PHA
is greater than about 500,000 Daltons and the molecular weight of
the second PHA is less than about 500,000 Daltons. However, in some
embodiments, the molecular weight can be adjusted. For example, in
some embodiments, a first PHA is subjected to a temperature
sufficient to reduce the molecular weight of the first PHA.
Thereafter, it can be combined with the second PHA. It shall be
appreciated that the second PHA could also optionally be exposed to
temperature in order to adjust its molecular weight. In some
embodiments, the molecular weight of the second PHA is greater than
about 800,000 Daltons. In certain embodiments, the molecular weight
of said second PHA is greater than about 1,000,000 Daltons. In some
embodiments, the molecular weights of the first and second PHA are
specifically tailored relative to one another, (e.g., a ratio of
1:2, 1:4, 1:6, 1:8, 1:10, etc.) in order to maximize the
alterations in functional characteristics.
[0092] In several embodiments, processes for increasing the
penetration depth of light in a liquid are provided. In one
embodiment, the process comprises the steps of (a) directing light
into a liquid medium in the form of a light path and (b) reducing
the density of liquid in the light path.
[0093] In several embodiments, the density of the liquid in the
light path is reduced by adding gas to said liquid in the light
path. In some embodiments, the gas is air, oxygen, methane, carbon
dioxide, nitrogen, and/or a combination thereof. In some
embodiments, the gas is simultaneously added along with the light.
In certain embodiments, the gas and the light are emitted or
injected into the liquid through a common material, such as a
permeable or semi-permeable membrane through which light and/or gas
traverse. In other embodiments, the light and the gas are added
separately. In such embodiments, customization of the addition is
possible. For example, gas can be added in pulses (e.g., on/off
sequences), continuously, or in bracketed time frames around the
addition of light. In some embodiments, the addition of light and
gas are coordinated to maximize the penetration of the light. For
example a burst of gas followed by a burst of light (or overlapping
to some degree) may advantageously increase the penetration of the
light.
[0094] In several embodiments, processes for modifying the pH in a
microorganism culture medium are provided. In one embodiment, the
process comprises the steps of: (a) providing a culture medium
comprising water and microorganisms, (b) adding a first source of
nitrogen to the medium to cause the microorganisms to metabolize
the nitrogen and thereby increase the concentration of either
hydroxyl ions or protons, respectively, in the medium, and (c)
adding a second source of nitrogen to the medium to cause the
microorganisms to metabolize the nitrogen and thereby increase the
concentration of either protons or hydroxyl ions, respectively, in
the medium. In other embodiments, a source of nitrogen is added to
the culture that increases the pH of the medium, wherein the
metabolism of the nitrogen source causes the pH of the medium to
decrease, thereby reducing or eliminating the need for an
additional pH adjustment step.
[0095] In several embodiments, low shear processes for adding gas
to a microorganism culture medium are provided. In one embodiment,
the process comprises the steps of: (a) providing a liquid medium
and a gas, (b) contacting the medium with the gas in a first
container to cause at least a portion of the gas to dissolve in the
medium, (c) providing a second container, and (d) transferring at
least a portion of the liquid comprising the gas within the first
container to the second container. In some embodiments, a mixer is
also provided, in order to dissolve a portion of the gas in the
medium. In some embodiments, the mixer is a pump or agitator or
high shear mixer. In some embodiments, the mixer comprises a
centrifugal pump. In still other embodiments, the gas itself
provides a mixing function. For example, the injection of gas into
a medium will result in gas bubbles, which, if released at the
bottom of a container comprising medium, will not only promote the
dissolution of gas into the medium, but mix the medium as the
bubbles rise.
[0096] In several embodiments, processes for injecting gas into a
pressurized microorganism culture vessel are provided. In one
embodiment, the process comprises the steps of: providing a vessel
comprising a medium comprising microorganisms, adding a gas into
the vessel that can be metabolized by the microorganisms, and
adjusting the flow rate of the gas into the vessel according to the
rate of change of pressure within the vessel. In some embodiments,
the gas is oxygen, methane, carbon dioxide, or combinations
thereof. Choice of the gas depends on the vessel used and the
culture within the vessel. In some embodiments, backpressure
monitoring allows for optimal gas injection for a given culture
(e.g., if certain cultures react more quickly to administration of
a gas and rapidly increase pressure, flow can be coordinately
reduced).
[0097] In one embodiment, a process for producing light in a liquid
medium is provided. In one embodiment, the process comprises the
steps of: a) providing a liquid, b) providing a light-emitting unit
or material comprising two conductive leads and a light-emitting
conjuncture between the conductive leads, or a material that will
emit light when contacted with electrons and c) inducing a voltage
in the liquid, thereby inducing the movement of electrons in the
conductive leads of the light-emitting unit or inducing electrons
to contact the material, thereby causing the light-emitting unit or
material to emit light. In some embodiments, the material is a
phosphor, phosphoric, and/or luminescent material, including an
electroluminescent phosphor.
[0098] In one embodiment, AC voltage is induced in a liquid by
inserting the two leads of a 115V AC power source into a liquid.
Without being bound by theory, it is believed that a liquid
carrying an AC voltage is capable of inducing the movement of
electrons into and through a light-emitting device suspended in the
liquid and not contacting the two 115V AC power source leads due to
the oscillating nature of electrons in an AC circuit, such that AC
voltage in a liquid causes electrons to fill conductive paths
connected to the liquid, in spite of the resistance of the
conductive paths relative to the liquid, and will oscillate as an
AC current in those conductive paths, thereby performing work,
e.g., generating light in a light emitting diode. As a non-limiting
example, light is produced in a liquid medium by a) placing a
light-emitting diode in a liquid comprising water and electrolytic
ions, and b) inducing an AC voltage in the liquid, wherein c) the
induction of AC voltage in the electrolytic liquid causes the
light-emitting diode to generate light.
[0099] Through experimentation, Applicant unexpectedly discovered
that one or multiple light emitting units will emit light in a
liquid when AC voltage is applied to the liquid and when the
light-emitting units are rated for voltages and amp draws
commensurate with available electrical energy. The production of
autotrophic microorganisms is fundamentally constrained by the
ability of light to penetrate through a liquid and thereby enable
photosynthesis. Prior to Applicant's invention as disclosed herein,
no methods were believed to be known to produce light in a liquid
through the utilization of light-emitting devices physically
unconnected to an electrical voltage source vis-a-vis solid
conductive material. In one embodiment, the utilization of one or
more, and preferably many, free-floating light-emitting units in an
electrically charged liquid comprising autotrophic microorganisms
enables a very high light transmission efficiency, wherein previous
light penetration constraints are largely overcome and high
autotrophic microorganism densities are fully enabled.
[0100] In several embodiments, a method for producing a
polyhydroxyalkanoate (PHA) in a microorganism culture is provided.
In some embodiments, the method comprises the steps of: a)
subjecting said culture to a growth period comprising exposing said
culture to growth conditions to cause said culture to reproduce,
(b) subjecting said culture to a polymerization period comprising
exposing said culture to polymerization conditions to cause said
culture to produce intracellular PHA, and (c) repeating step (a)
and then second step (b) two or more times.
[0101] In some embodiments the growth period comprises a period in
which the culture reproduces or otherwise produces biomass and/or
reproduces. In some embodiments, the polymerization period
comprises a period in which the culture synthesizes PHA. In some
embodiments, the growth period and the polymerization period are
induced by the culture media (e.g., the extracellular media around
the culture). In some embodiments, alteration in the media
conditions induce a transition (partial or complete) between growth
and polymerization periods. In some embodiments, a culture is
cycled between growth and polymerization periods two, three, four,
or more times, in order to produce PHA and then reproduce biomass,
which is subsequently used to generate additional PHA.
[0102] In some embodiments, the culture is exposed to non-sterile
conditions. In certain such embodiments, input carbon is
non-sterile. However, in some embodiments, sterile conditions
exist. In some embodiments, the culture is dynamic over time in
that it may be exposed to extraneous microorganisms. In certain
embodiments, this is due to a non-sterile culture environment. In
some embodiments, extraneous microorganisms potentiate the
production of PHA.
[0103] In several embodiments, a process for the reduction of
pigmentation in a microorganism culture is provided. In one
embodiment, the process comprises the steps of: providing a medium
comprising a microorganism culture comprising dissolved oxygen, and
increasing the concentration of dissolved oxygen over successive
periods to select for light-colored or low-level pigmentation
microorganisms.
[0104] PHA, while able to be treated post-production to reduce
pigmentation, is less expensive to produce when lower levels of
pigmentation exist. Some microorganisms are more pigmented than
others, and therefore in several embodiments, selection against the
more pigmented microorganisms results in a less pigmented PHA,
which reduces production costs. In some embodiments, the
microorganisms cultured and selected for are methanotrophic
microorganisms. By manipulating the culture conditions, which
benefit certain varieties of microorganisms, a less pigmented
culture (and hence a less pigmented PHA) result. In some
embodiments, increases in concentration of dissolved oxygen over
periods ranging from 1-3 hours, 3-5 hours, 5-7 hours, 7-10 hours,
10-15 hours, or 15-24 hours are used to select for less pigmented
microorganisms.
[0105] In several embodiments, a process for the conversion of a
gas into a polyhydroxyalkanoate (PHA) is provided, wherein the
process comprises the steps of: a) providing i) a first gas and ii)
a culture of microorganisms, b) contacting the first gas with the
culture to cause the culture to convert the first gas into a second
gas c) contacting the second gas with the culture, and d) causing
the culture to use the second gas to produce PHA.
[0106] In some embodiments, the microorganisms are autotrophic,
methanogenic, heterotrophic, or combinations thereof.
[0107] In one embodiment the first gas is carbon dioxide. In one
embodiment the first gas is oxygen. In one embodiment the first gas
is methane. In one embodiment the first gas is a volatile organic
compound.
[0108] In one embodiment the second gas is carbon dioxide. In one
embodiment the second gas is oxygen. In one embodiment the second
gas is methane. In one embodiment the second gas is a volatile
organic compound.
[0109] It shall be appreciated that the selection of the first and
the second gas is based on the type of microorganism or
microorganisms being cultured.
BRIEF DESCRIPTION OF THE DRAWING
[0110] FIG. 1 is a block flow diagram comprising the steps of:
microorganism fermentation and PHA synthesis, PHA-containing
biomass removal, PHA-reduced biomass and isolated PHA production,
PHA-reduced biomass recycling and fermentation, and isolated PHA
purification.
DETAILED DESCRIPTION
[0111] While PHAs have significant environmental advantages
compared to fossil fuel-based plastics, the cost of PHA production
is generally viewed as a significant limitation to the industrial
production and commercial adoption of PHAs. Generally, the overall
cost of PHA production is determined by three major inputs: 1)
carbon, 2) chemicals, and 3) energy. Accordingly, efforts to reduce
the cost of PHA production must address one or more of these areas,
specifically by: i) reducing carbon input costs, ii) increasing
carbon-to-PHA yields, iii) reducing the volume of chemicals
required for PHA production, and/or iv) increasing energy-to-PHA
yields.
[0112] As discussed above, food crop derived sugars in genetically
engineered microorganism-based aqueous fermentation systems are
widely regarded as the most carbon, chemical, energy, and, thus,
cost efficient PHA production method. Despite these efficiencies,
sugar-based PHA production remains many times more expensive than
fossil fuel-based plastics production. Attempts to reduce the
carbon input cost of the PHA production process, by utilizing
carbon-containing industrial off-gases, such as carbon dioxide and
methane, have been previously limited by technical challenges and
stoichiometric limitations that render the gas-to-PHA production
process significantly more energy and chemical intensive, and thus
more costly, than the food crop-based PHA production process.
[0113] Specifically, these technical challenges and stoichiometric
limitations include: low mass transfer rates, low microorganism
growth rates, extended polymerization times, low cell densities,
high oxygen demand (relative to solid substrates), and low PHA
cellular inclusion concentrations. Whereas sugar-based fermentation
systems have the ability to generate high cellular densities and
PHA inclusion concentrations, carbon-containing gas-based
fermentation processes typically cannot, based on fundamental cell
morphology and mass transfer constraints, generate cellular and PHA
densities exceeding 10-30% of densities possible in sugar-based
processes. As a result, the ratio of energy-to-PHA required to
carry out upstream carbon injection, oxygen injection, system
cooling, and culture mixing, as well as downstream PHA
purification, significantly exceeds the energy-to-PHA ratio
required for sugar-based PHA production methods, thereby rendering
the emissions-based process uncompetitive when compared to both
petroleum-based plastics and sugar-based PHAs.
[0114] Several embodiments of the present invention therefore
relate to a novel method for the production of PHA using
carbon-containing gases as a source of carbon, wherein the energy
input-to-PHA production ratio, carbon input-to-PHA production
ratio, and cost efficiency of the process is significantly improved
over previous gas-based PHA production processes.
[0115] In several embodiments, this process may be accomplished by
a) culturing a first microorganism culture capable of metabolizing
the carbon within both a carbon-containing gas and biomass, or a
derivative thereof, b) manipulating the conditions of the culture
to cause the culture to produce PHA-containing biomass, c) removing
a portion of the PHA-containing biomass; d) extracting at least a
portion of the PHA within the removed PHA-containing biomass to
create substantially isolated PHA and substantially PHA-reduced
biomass, e) purifying the isolated PHA, and f) returning the
PHA-reduced biomass to the microorganism culture to cause the
microorganism culture to metabolize the carbon within the
PHA-reduced biomass into PHA.
[0116] According to some embodiments, the steps of this process are
as follows: (a) providing a microorganism culture comprising
biomass and PHA; (b) removing a portion of the PHA-containing
biomass from the culture, and extracting PHA from the removed
PHA-containing biomass to produce isolated PHA and PHA-reduced
biomass; (c) purifying the isolated PHA, and (d) returning the
PHA-reduced biomass to be mixed with the culture to cause the
culture to convert the carbon within the PHA-reduced biomass into
PHA. Each of the above recited steps in the process are discussed
in more detail below.
Providing a Microorganism Culture Comprising Biomass and PHA
[0117] The terms "microorganism", "microorganisms", "culture",
"cultures", and "microorganism cultures", as used herein, shall be
given their ordinary meaning and shall include, but not be limited
to, a single microorganism and/or consortium of microorganisms,
including, among others, genetically-engineered bacteria, fungi,
algae, and/or yeast. In some embodiments, microorganisms are
naturally occurring and in some embodiments microorganisms are
genetically-engineered. In some embodiments, both naturally
occurring and genetically-engineered microorganisms are used. In
some embodiments, a mixed culture of microorganisms may be used. In
some embodiments, microorganisms or cultures shall include a
microorganism metabolism system, including the interactions and/or
multiple functions of multiple cultures in one or more
conditions.
[0118] The terms "biomass" and "biomass material" shall be given
their ordinary meaning and shall include, but not be limited to,
microorganism-derived material, including intracellular, cellular,
and/or extracellular material, such materials including, but not
limited to, a polymer or polymers, amino acids, nucleic acids,
carbohydrates, lipids, sugars, PHA, volatile fatty acids,
chemicals, gases, such as carbon dioxide, methane, volatile organic
acids, and oxygen, and/or metabolic derivatives, intermediaries,
and/or end-products. In several embodiments, biomass is dried or
substantially dried.
[0119] In some embodiments, the biomass contains less than about
99% water. In other embodiments, the biomass contains between about
99% to about 75% water, including about 95%, 90%, 85%, and 80%. In
some embodiments, the biomass contains between about 75% and about
25% water, including 75%-65%, 65%-55%, 55%-45%, 45%-35%, 35%-25%,
and overlapping ranges thereof. In additional embodiments, the
biomass contains from about 25% water to less than about 0.1%
water, including 25%-20%, 20%-15%, 15%-10%, 10%-5%, 5%-1%, 1%-0.1%,
and overlapping ranges thereof. In still other embodiments, the
biomass contains no detectable amount of water. Depending on the
embodiment, water is removed from the biomass by one or more of
freeze drying, spray drying, fluid bed drying, ribbon drying,
flocculation, pressing, filtration, and/or centrifugation. In some
embodiments, the biomass may be mixed with one or more chemicals,
such as methylene chloride, acetone, methanol, and/or ethanol, at
various concentrations. In other embodiments, the biomass may be
processed through homogenization, heat treatment, pH treatment,
enzyme treatment, solvent treatment, spray drying, freeze drying,
sonication, or microwave treatment. As used herein, the term
"PHA-reduced biomass" shall be given its ordinary meaning and shall
mean any biomass wherein at least a portion of PHA has been removed
from the biomass through a PHA extraction process. As used herein,
the term "PHA-containing biomass" shall be given its ordinary
meaning and shall mean any biomass wherein at least a portion of
the biomass is PHA.
[0120] Microorganism cultures useful for the invention described
herein include a single strain, and/or a consortium of strains,
which are individually and/or collectively capable of using carbon
containing gases and biomass, including PHA-reduced biomass, as a
source of carbon for the production of biomass and PHA. In some
embodiments, a microorganism culture according to several
embodiments, comprises a microorganism culture that utilizes
PHA-reduced biomass, or any derivative thereof, including
methanotrophic microorganisms, anaerobic digestion cultures, and
other heterotrophic microorganisms, as a source of carbon for the
production of biomass, or metabolic derivatives including, and in
particular, the production of PHA, protein, methane, and/or carbon
dioxide (herein, "biomass-utilizing microorganisms"). As used
herein, the terms "microorganism", "culture", "microorganism
culture," "microorganism system," "microorganism consortium," and
"consortium of microorganisms" are used interchangeably.
Additionally, any of these terms may refer to one, two, three, or
more microorganism cultures and/or strains, including a
microorganism system that is collectively capable of carrying out a
complex metabolic function (e.g., conversion of PHA-reduced biomass
to methane, carbon dioxide, protein, and/or PHA). In several
embodiments, the microorganism culture comprises of a consortium of
carbon-containing gas-utilizing microorganisms and a consortium of
biomass-utilizing microorganisms. In some embodiments, the gases
metabolized by such cultures comprise methane, carbon dioxide,
and/or a combination thereof.
[0121] In some embodiments, the microorganism culture comprises a
consortium of acidogenic, acetogenic, methanogenic, methanotrophic,
and/or autotrophic microorganisms in one or more individual
bioreactors. As such, in some embodiments, the cultures are grown
in one or more distinct culture conditions. In some embodiments,
the conditions are either aerobic or anaerobic conditions. In some
embodiments, culture conditions are varied over time (e.g.
initially aerobic with a transition to anaerobic, or vice versa).
As used herein, the term "bioreactor" shall be given its ordinary
meaning and shall also refer to a tank, vessel, or any container or
device suitable for growth and culturing of microorganisms.
[0122] In some embodiments, the microorganism culture is contained
within a single vessel, wherein the steps of converting PHA-reduced
biomass to biomass, converting biomass to PHA, and converting
carbon-containing gases to biomass and/or PHA occur simultaneously
or sequentially.
[0123] In other embodiments, the microorganism culture is contained
within multiple vessels, which are designed to carry out specific
and unique functions. For example, one embodiment includes the
steps of (a) converting PHA-reduced biomass to PHA-reduced
biomass-derived materials such volatile organic acids, methane,
and/or carbon dioxide, which is carried out in a first vessel and
(b) synthesizing PHA from PHA-reduced biomass-derived materials
which is carried out in a second, separate tank under independent
conditions. In some embodiments, one or more of the tanks is an
anaerobic digestion tank and one or more other tank is an aerobic
fermentation tank.
[0124] As used herein, the term "gas-utilizing microorganisms"
shall be given its ordinary meaning and shall refer to
microorganisms capable of utilizing gases containing carbon for the
production of biomass, including the production of PHA. Similarly,
the terms "methanotrophic microorganisms" and "methane-utilizing
microorganisms" shall be given their ordinary meanings and shall
refer to microorganisms capable of utilizing methane as a source of
carbon for the production of biomass. Further, the terms
"Autotrophic microorganisms" and "carbon dioxide-utilizing
microorganisms" shall be given their ordinary meaning and shall
refer to microorganisms capable of utilizing carbon dioxide as a
source of carbon for the production of biomass, including
microorganisms that utilize natural and/or synthetic sources of
light to carry out the metabolism of carbon dioxide into biomass.
The term "heterotrophic microorganisms", as used herein, shall be
given its ordinary meaning and shall include methanotrophic,
methanogenic, acidogenic, acetogenic and biomass-utilizing
microorganisms, including microorganisms that convert sugar,
volatile fatty acids, or other carbon substrates to biomass. The
term "methanogenic microorganisms" shall be given its ordinary
meaning and shall refer to microorganisms that convert biomass to
methane, including the consortium of microorganisms required to
carry out such a process, including, but not limited to, acidogenic
and acetogenic microorganisms.
[0125] As discussed herein, in several embodiments,
carbon-containing gases are used as a source of carbon by
microorganism cultures. In some embodiments, other sources of
carbon are used (e.g., PHA-reduced biomass), either alone or in
combination with carbon-containing gases. In some embodiments, the
carbon-containing gases used include, but are not limited to,
carbon dioxide, methane, ethane, butane, propane, benzene, xylene,
acetone, methylene chloride, chloroform, volatile organic
compounds, hydrocarbons, and/or combinations thereof. The source of
the carbon-containing gases depends on the embodiment. For example,
carbon-containing gas sources used in some embodiments include
landfills, wastewater treatment plants, anaerobic metabolism, power
production facilities or equipment, agricultural digesters, oil
refineries, natural gas refineries, cement production facilities,
and/or anaerobic organic material digesters, including both solid
and liquid material digesters.
[0126] In several embodiments described herein, microorganisms may
include, but are not limited to, yeast, fungi, algae, and bacteria
(including combinations thereof). Suitable yeasts include, but are
not limited to, species from the genera Candida, Hansenula,
Torulopsis, Saccharomyces, Pichia, 1-Debaryomyces, Lipomyces,
Cryptococcus, Nematospora, and Brettanomyces. Suitable genera
include Candida, Hansenula, Torulopsis, Pichia, and Saccharomyces.
Examples of suitable species include, but are not limited to:
Candida boidinii, Candida mycoderma, Candida utilis, Candida
stellatoidea, Candida robusta, Candida claussenii, Candida rugosa,
Brettanomyces petrophilium, Hansenula minuta, Hansenula satumus,
Hansenula californica, Hansenula mrakii, Hansenula silvicola,
Hansenula polymorpha, Hansenula wickerhamii, Hansenula capsulata,
Hansenula glucozyma, Hansenula henricii, Hansenula nonfermentans,
Hansenula philodendra, Torulopsis candida, Torulopsis bolmii,
Torulopsis versatilis, Torulopsis glabrata, Torulopsis molishiana,
Torulopsis nemodendra, Torulopsis nitratophila, Torulopsis pinus,
Pichia farinosa, Pichia polymorpha, Pichia membranaefaciens, Pichia
pinus, Pichia pastoris, Pichia trehalophila, Saccharomyces
cerevisiae, Saccharomyces fragilis, Saccharomyces rosei,
Saccharomyces acidifaciens, Saccharomyces elegans, Saccharomyces
rouxii, Saccharomyces lactis, and/or Saccharomyces fractum.
[0127] Suitable bacteria include, but are not limited to, species
from the genera Bacillus, Mycobacterium, Actinomyces, Nocardia,
Pseudomonas, Methanomonas, Protaminobacter, Methylococcus,
Arthrobacter, Methylomonas, Brevibacterium, Acetobacter,
Methylomonas, Brevibacterium, Acetobacter, Micrococcus,
Rhodopseudomonas, Corynebacterium, Rhodopseudomonas,
Microbacterium, Achromobacter, Methylobacter, Methylosinus, and
Methylocystis. Preferred genera include Bacillus, Pseudomonas,
Protaminobacter, Micrococcus, Arthrobacter and/or Corynebacterium.
Examples of suitable species include, but are not limited to:
Bacillus subtilus, Bacillus cereus, Bacillus aureus, Bacillus
acidi, Bacillus urici, Bacillus coagulans, Bacillus mycoides,
Bacillus circulans, Bacillus megaterium, Bacillus licheniformis,
Pseudomonas ligustri, Pseudomonas orvilla, Pseudomonas methanica,
Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas
oleovorans, Pseudomonas putida, Pseudomonas boreopolis, Pseudomonas
pyocyanea, Pseudomonas methylphilus, Pseudomonas brevis,
Pseudomonas acidovorans, Pseudomonas methanoloxidans, Pseudomonas
aerogenes, Protaminobacter ruber, Corynebacterium simplex,
Corynebacterium hydrocarbooxydans, Corynebacterium alkanum,
Corynebacterium oleophilus, Corynebacterium hydrocarboclastus,
Corynebacterium glutamicum, Corynebacterium viscosus,
Corynebacterium dioxydans, Corynebacterium alkanum, Micrococcus
cerificans, Micrococcus rhodius, Arthrobacter rufescens,
Arthrobacter parafficum, Arthrobacter citreus, Methanomonas
methanica, Methanomonas methanooxidans, Methylomonas agile,
Methylomonas albus, Methylomonas rubrum, Methylomonas methanolica,
Mycobacterium rhodochrous, Mycobacterium phlei, Mycobacterium
brevicale, Nocardia salmonicolor, Nocardia minimus, Nocardia
corallina, Nocardia butanica, Rhodopseudomonas capsulatus,
Microbacterium ammoniaphilum, Archromobacter coagulans,
Brevibacterium butanicum, Brevibacterium roseum, Brevibacterium
flavum, Brevibacterium lactofermentum, Brevibacterium
paraffinolyticum, Brevibacterium ketoglutamicum, and/or
Brevibacterium insectiphilium.
[0128] In several embodiments, more than one type or species of
microorganism is used. For example, in some embodiments, both algae
and bacteria are used. In some embodiments, several species of
yeast, algae, fungi, and/or bacteria are used. In some embodiments,
a single yeast, algae, fungi, and/or bacteria species is used. In
some embodiments, a consortium of cyanobacteria is used. In some
embodiments, a consortium of methanotrophic microorganisms is used.
In still additional embodiments, a consortium of both
methanotrophic bacteria and cyanobacteria are used. In several
embodiments, methanotrophic, heterotrophic, methanogenic, and/or
autotrophic microorganisms are used.
[0129] In several embodiments of the invention, the microorganism
culture comprises a consortium of methanotrophic, autotrophic,
and/or heterotrophic microorganisms, wherein methane and/or carbon
dioxide is individually, interchangeably, or simultaneously
utilized for the production of biomass. In some embodiments,
PHA-reduced biomass is used as a source of carbon by heterotrophic,
autotrophic, and/or methanotrophic microorganisms. In several
embodiments of the invention, the microorganism culture comprises
methanotrophic microorganisms, cyanobacteria, and
non-methanotrophic heterotrophic microorganisms, wherein methane
and carbon dioxide are continuously utilized as sources of carbon
for the production of biomass and PHA.
[0130] In some embodiments, microorganisms are employed in a
non-sterile, open, and/or mixed environment. In other embodiments,
microorganisms are employed in a sterile and/or controlled
environment.
[0131] The terms "PHA", "PHAs", and "polyhydroxyalkanoate", as used
herein, shall be given their ordinary meaning and shall include,
but not be limited to, polymers generated by microorganisms as
energy and/or carbon storage vehicles; biodegradable and
biocompatible polymers that can be used as alternatives to
petrochemical-based plastics such as polypropylene, polyethylene,
and polystyrene; polymers produced by bacterial fermentation of
sugars, lipids, or gases; and/or thermoplastic or elastomeric
materials derived from microorganisms. PHAs include, but are not
limited to, polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV),
polyhydroxybutyrate-covalerate (PHB/V), and polyhydroxyhexanoate
(PHHx), as well as both short chain length (SCL), medium chain
length (MCL), and long chain length (LCL) PHAs.
[0132] The terms "growth-culture medium", "growth medium",
"growth-culture media", "medium", and "media", as used herein shall
be given their ordinary meaning and shall also refer to materials
affecting the growth, metabolism, PHA synthesis, and/or
reproductive activities of microorganisms. Non-limiting examples of
growth-culture media used in several embodiments include a mineral
salts medium, which may comprise water, nitrogen, vitamins, iron,
phosphorus, magnesium, and various other nutrients suitable to
effect, support, alter, modify, control, constrain, and/or
otherwise influence the metabolism and metabolic orientation of
microorganisms. A growth-culture medium may comprise water filled
with a range of mineral salts. For example, each liter of a liquid
growth-culture medium may be comprised of about 0.7-1.5 g
KH.sub.2PO.sub.4, 0.7-1.5 g K.sub.2HPO.sub.4, 0.7-1.5 g KNO.sub.3,
0.7-1.5 g NaCl, 0.1-0.3 g MgSO.sub.4, 24-28 mg
CaCl.sub.2*2H.sub.2O, 5.0-5.4 mg EDTA Na.sub.4(H.sub.2O).sub.2,
1.3-1.7 mg FeCl.sub.2*4H.sub.2O, 0.10-0.14 mg CoCl.sub.2*6H.sub.2O,
0.08-1.12 mg MnCl.sub.2*2H.sub.2O, 0.06-0.08 mg ZnCl.sub.2,
0.05-0.07 mg H.sub.3BO.sub.3, 0.023-0.027 mg NiCl.sub.2*6H.sub.2O,
0.023-0.027 mg NaMoO.sub.4*2H.sub.2O, and 0.011-0.019 mg
CuCl.sub.2*2H.sub.2O. A growth-culture medium can be of any form,
including a liquid, semi-liquid, gelatinous, gaseous, foam, or
solid substrate.
[0133] In several embodiments of the invention, a microorganism
culture is produced in a liquid growth medium, wherein carbon
dioxide and methane are utilized as a gaseous source of carbon for
the production of methanotrophic and/or autotrophic biomass. In
some embodiments, PHA-reduced methanotrophic and/or PHA-reduced
autotrophic biomass is utilized as a source of carbon for the
production of heterotrophic biomass and heterotrophically-produced
PHA. In some embodiments, the growth medium is manipulated to
effect the growth, reproduction, and PHA synthesis of the
microorganism culture. Methods for the production of methanotrophic
microorganisms are disclosed in the art, and are described by
Herrema, et al., in U.S. Pat. No. 7,579,176, which is hereby
incorporated by reference in its entirety. Methods for the
production of cyanobacteria are described by Lee, et al.
("High-density algal photobioreactors using light-emitting diodes,"
Biotechnology and Bioengineering, Vol. 44, Issue 10, pp.
1161-1167), which is hereby incorporated by reference in its
entirety. Methods for the production of methane from biomass are
described by Deublein, et al. ("Biogas from Waste and Renewable
Resources, WILEY-VCH Verlag GmbH & Co. KgaA, 2008), which is
hereby incorporated by reference in its entirety. In some
embodiments, PHA synthesis may be effected through the manipulation
of one of more elements of the culture medium, including through
the reduction, increase, or relative change in either the total or
bioavailable concentration of one or more of the following
elements: nitrogen, phosphorus, oxygen, methane, carbon dioxide,
magnesium, potassium, iron, copper, sulfate, manganese, calcium,
chlorine, boron, zinc, aluminum, nickel, and/or sodium. Methods for
the production of PHA are described by Herrema, et al., in U.S.
Pat. No. 7,579,176.
[0134] In several embodiments of the invention, methanol is added
to a culture of methanotrophic microorganisms utilizing a closed
loop recycling gas stream comprising methane. In some embodiments,
methanotrophic microorganisms are enabled to grow under conditions
of, and consume, very low concentrations of methane by co-utilizing
methanol as a carbon substrate. In the past, the growth of
methanotrophic microorganisms was significantly reduced under low
methane concentrations due to, among other things, low mass
transfer rates. In some embodiments, by the addition of methanol in
a closed loop gas recycling system, it is possible to effect the
substantially complete elimination of methane by methanotrophic
microorganisms.
[0135] In several embodiments of the invention, the diffusion of
light is increased in a liquid growth culture media by reducing the
density of the liquid in a light path. In some embodiments the
culture comprises autotrophic microorganisms. In some embodiments,
the application of gas bubbles into the media decreases the
relative solids density of the light path, thus enabling an
increased diffusion of light into a liquid culture media from a
given light intensity energy.
[0136] In several embodiments of the invention, a series of
submerged light rods are placed into a liquid culture to manipulate
or adjust the culture conditions. In some embodiments, the culture
comprises autotrophic microorganisms. In some embodiments, the
light rods function to diffuse light, diffuse gas, act as static or
dynamic mixers, assist in the circulation of a liquid culture
media, and/or facilitate heat exchange through the circulation of a
gas, liquid, and/or combination thereof.
[0137] Traditionally, pH control in a microorganism growth system
is difficult and/or costly to maintain. In some embodiments, pH is
controlled by varying the nitrogen source supplied to a
microorganism growth system between pH-increasing and pH-reducing
nitrogen sources, e.g., NO.sub.3.sup.- and NH.sub.3.sup.+,
respectively. In some embodiments, nitrogen sources are utilized
that do not significantly affect the pH of the system, including,
when applicable, complex nitrogen sources such as biomass. In
additional embodiments, a closed loop system is employed to reduce
changes in pH. In some embodiments, respiration-generated carbon
dioxide counterbalances increases in pH caused by the utilization
of pH-increasing nitrogen sources, such as nitrates.
[0138] A number of methods are known for the induction of gas into
liquid, including static mixing, ejector mixing, propeller mixing,
and/or a combination thereof. Simultaneously, it is also known that
shear can be highly detrimental to microorganism growth, and can
often impede or permanently deactivate microorganism metabolism.
Thus, mass transfer in a gas-based system is often limited by the
need to counterbalance sufficient mixing with shear considerations.
In several embodiments of the invention, a vessel comprising liquid
culture media is mixed with a gas, e.g. methane, under relatively
high shear conditions, and then subsequently transferred to a
vessel comprising liquid culture media maintained under relatively
low shear conditions. In some embodiments, microorganism growth is
primarily induced in the low shear vessel. In some embodiments,
high gas transfer rates are effected in the first high shear vessel
by mixing while performed in the low shear vessel by gaseous
diffusion.
[0139] In another embodiment, a closed loop gas recycling system is
maintained, wherein a vessel comprising gas-utilizing
microorganisms is supplied with gas, wherein the gas is utilized by
gas-utilizing microorganisms, and wherein the rate at which gas is
added to the system is determined by the rate at which the pressure
in the vessel changes in accordance with the conversion of gases
into metabolic derivatives (such as biomass, carbon dioxide, and
water). For example, a vessel containing methane-utilizing
microorganisms may be pressurized to 60 psi with a combination of
methane and oxygen; as the pressure in the system drops in
accordance with the metabolism of the methane-utilizing
microorganisms, additional methane and oxygen is added to the
system such that the pressure of the vessel remains at 60 psi. It
shall be appreciated that, in certain embodiments, higher or lower
pressures are maintained. In some embodiments, the system is
periodically flushed to remove carbon dioxide. In some embodiments,
autotrophic microorganisms and a light injection system may be
added to the system in order to convert carbon dioxide into
additional oxygen, thereby substantially reducing or eliminating
the need to flush the system and/or introduce oxygen.
[0140] In several embodiments, PHA synthesis is induced in a
microorganism culture comprising methane-utilizing, heterotrophic,
and/or carbon dioxide-utilizing microorganisms wherein a PHA
inclusion concentration (by dry biomass weight) is generated of
between about 0.01% and 95%. In some embodiments, the inclusion
concentration is between 25% and 80%, including 25-35%, 35% to 50%,
50% to 65%, 65% to 80%, and overlapping ranges thereof. In some
embodiments, the inclusion concentration is between 0.01% and 55%,
including, 0.01% to 1%, 1% to 5%, 5% to 10%, 10% to 15%, 15% to
20%, 20%, to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%,
45% to 50%, 50% to 55%, and overlapping ranges thereof. In some
embodiments, PHA synthesis is induced in a methanotrophic,
heterotrophic, and/or autotrophic microorganism culture wherein a
PHA inclusion concentration (by dry biomass weight) is generated of
between 20% and 80%, between 30% and 70%, between 40% and 60%,
between 50% and 70%, including 50% to 55%, 55 to 60%, 60% to 65%,
65% to 70%, and overlapping ranges thereof. In some embodiments of
the invention, PHA synthesis is induced in microorganism culture
comprising methanotrophic, autotrophic, and heterotrophic
microorganisms, wherein an average PHA inclusion concentration (by
dry biomass weight) is greater than 5%, greater than 20%, greater
than 40%, greater than 65%, or greater than 70% by dry cell
weight.
[0141] In some embodiments, the growth culture media is manipulated
to induce both i) microorganism growth and ii) PHA synthesis within
one or more open, non-sterile, or sterile vessels using a
feast-famine culture regime. In some embodiments, the
microorganisms are subject to successive alternating periods of
nutrient/carbon availability and nutrient/carbon unavailability to
encourage the reproductive success of microorganisms that are
capable of synthesizing PHA, particularly at high inclusion
concentrations. Feast-famine regimes useful for the selection of
PHA producing microorganisms, including PHA-producing
methanotrophic microorganisms, over microorganisms that either
cannot produce PHA, produce PHA slowly, or produce PHA at
relatively low concentrations are described in the art (Frigon, et
al., "rRNA and Poly-Hydroxybutyrate Dynamics in Bioreactors
Subjected to Feast and Famine Cycles," Applied and Environmental
Microbiology, Apr. 2006, p. 2322-2330; Muller, et al., "Adaptive
responses of Ralstonia eutropha to feast and famine conditions
analysed by flow cytometry," J Biotechnol. 1999 October 8;
75(2-3):81-97; Reis, et al., "Production of polyhydroxyalkanoates
by mixed microbial cultures," Bioprocess and Biosystems
Engineering, Volume 25, Number 6, 377-385, DOI:
10.1007/s00449-003-0322-4.)
[0142] In some embodiments of the invention, the classical
feast-famine regime is modified to reduce PHA losses. Specifically,
in the past, feast-famine regimes were thought to be effective by
passing a microorganism culture through a period wherein carbon or
nutrients were unavailable or relatively limited for metabolism,
thereby forcing the culture to accumulate and/or consume
intracellular PHA as a source of carbon to survive, and thereby
selecting for microorganisms with the capacity to synthesize and
store PHA. Applicant has surprisingly discovered that, some
microorganisms with higher concentrations of intracellular PHA
reproduce more efficiently than microorganisms with lower
concentrations of intracellular PHA in periods of carbon
availability and nutrient balance. Thus, in one embodiment, a novel
PHA production regime is employed in one or more vessel wherein
microorganisms are subjected to two successive and recurring
phases: 1) growth, wherein carbon and nutrient availability is
optimized for reproduction, and 2) PHA synthesis, wherein carbon is
available in excess, and one or more nutrient is reduced or
increased relative to the growth period to induce PHA synthesis. In
some embodiments, a fraction of the vessel media is removed for
downstream PHA extraction and processing after the PHA synthesis
period, and that fraction is replaced with lower cell density
media, which simultaneously returns carbon and nutrient
concentrations to reproductively favorable levels, e.g., the growth
phase or growth conditions, and causes microorganisms to enter into
a reproductive phase without consuming significant portions of
intracellular PHA. As such, efficient PHA producing microorganisms
selectively reproduce over inefficient or non-PHA producing
microorganisms. As a result, some embodiments, i) increase the
speed of the microorganism selection process by removing the PHA
consumption step typical to previous feast famine models and ii)
reduce the loss of PHA to cellular metabolism. According to such
embodiments, the feast famine model is converted to a
feast-polymerization-feast process. In several embodiments
methanotrophic, autotrophic, and/or heterotrophic cultures are used
in the feast-polymerization-feast process.
Removing a Portion of the PHA-Containing Biomass from the Culture
and Extracting PHA from the Removed PHA-Containing Biomass to
Produce Isolated PHA and PHA-Reduced Biomass
[0143] In several embodiments, following the production of a
microorganism culture comprising biomass and PHA (discussed above),
at least a portion of the PHA-containing biomass is removed from
the culture. In several embodiments, a portion ranging from 20% to
80% of the PHA-containing biomass is removed, including 30%-70%,
40% to 60%, 45% to 55%, and overlapping ranges thereof. Removal of
PHA-containing biomass may be performed by a number of methods,
including centrifugation, filtration, density separation,
flocculation, agglomeration, spray drying, or other separation
technique. In some embodiments, dewatering (e.g., by
centrifugation) results in a biomass having a desirable water
content that facilitates downstream processing of the biomass. For
example, in some embodiments, centrifugation of the PHA-containing
biomass reduces the amount of culture media (increases the relative
biomass concentration) to a concentration range between about 100
and 500 grams of biomass per liter of culture media. In some
embodiments, the concentration is of the biomass is adjusted to
about 100 to 200 g/L, 200 to 300 g/L, 300 to 400 g/L, 400 to 500
g/L, and overlapping ranges thereof. Advantageously, such an
approach also produces, as an effective by-product, clarified
culture media that can be optionally treated, measured, or recycled
into one or more culture vessels.
[0144] In some embodiments, after a portion of the PHA-containing
biomass is removed from the culture, PHA is extracted from the
removed PHA-containing biomass to produce isolated PHA and
PHA-reduced biomass.
[0145] As used herein, the terms "extraction" and "PHA extraction"
shall be given their ordinary meaning and shall be used
interchangeably to describe the removal and/or separation of PHA
from biomass. PHAs may be extracted from biomass by several
processes, including, but not limited to, the use of chemicals,
mechanical means, solvents, and enzymes. These processes include
the use of: i) solvents, such as acetone, ethanol, methanol,
methylene chloride, and dichloroethane, with and/or without the
application of pressure and/or elevated temperatures, ii)
supercritical carbon dioxide, iii) enzymes, such as protease, iv)
surfactants, v) pH adjustment, including the protonic or
hydroxide-based dissolution of non-PHA biomass, and/or vi)
hypochlorite to dissolve non-PHA biomass, including the use of
hypochlorite in conjunction with a solvent, such as methylene
chloride. In some embodiments of the invention, PHA is extracted by
solvent extraction from a PHA-containing biomass comprising
gas-utilizing microorganisms and/or biomass-utilizing
microorganisms to produce isolated PHA and PHA-reduced biomass. In
some solvent-based embodiments, solvents suitable for dissolving
the PHA are used, including carbon dioxide, acetone, methylene
chloride, chloroform, water, ethanol, and methanol. In some
embodiments, particular ratios of solvent to PHA provide optimal
dissolution of PHA from the culture, and therefore lead to improved
extraction and isolation efficiency and yield. For example, in some
embodiments, ratios of solvent to PHA (in grams) of about 500:1 are
used. In some embodiments, ratios of about 0.01:1 are used. In some
embodiments, ratios ranging from between about 500:1 and 0.01:1 are
used, such as 0.05:1, 1.0:1, 1.5:1, 20:1, 250:1, 300:1, 350:1,
400:1, or 450:1.
[0146] As discussed above, changes in temperature and/or pressure
may also be used to facilitate the extraction of PHA from the
PHA-containing biomass. In some embodiments, the extraction solvent
chosen dictates the limits of temperature, pressure, and/or
incubation times that are used. In some embodiments, solvent is
combined with PHA-containing biomass and incubated for several
minutes up to several hours. For example, in some embodiments,
incubation is for about 10 minutes, while in other embodiments,
overnight incubation times are used. In some embodiments,
incubation times range from 30 minutes to about 1 hour, about 1
hour to about 2 hours, about 2 hours to about 4 hours, about 4
hours to about 6 hours, about 6 hours to about 8 hours, about 8
hours to about 10 hours, and from about 10 hours to overnight.
Choice of incubation time is determined by solvent, culture density
(e.g., number of microorganisms), type of organisms, expected PHA
yield, and other similar factors.
[0147] Incubation temperature is also tailored to the
characteristics of a given culture. Incubation temperatures can
range from below room temperature to elevated temperatures of up to
about 150.degree. C. or about 200.degree. C. For example, depending
the solvent and other variables of the culture, temperatures are
used that range from about 10.degree. C. to 25.degree. C., from
about 25.degree. C. to about 40.degree. C., from about 40.degree.
C. to about 55.degree. C., from about 55.degree. C. to about
60.degree. C., from about 60.degree. C. to about 75.degree. C.,
from about 75.degree. C. to about 90.degree. C., from about
90.degree. C. to about 105.degree. C., from about 105.degree. C. to
about 120.degree. C., from about 120.degree. C. to about
135.degree. C., from about 135.degree. C. to about 150.degree. C.,
from about 150.degree. C. to about 200.degree. C., and overlapping
ranges thereof.
[0148] As can be appreciated, if changes in temperature are made to
a culture in a closed vessel, changes in pressure result. In some
embodiments, increased pressure provides a shearing effect that
aids in the liberation of PHA from the microorganisms. In some
embodiments, pressure is regulated within a particular range. For
example, in some embodiments, pressure of the reaction of the
PHA-containing biomass with solvent occurs between about 40 and 200
psi, including about 50 to 60 psi, 60 to 70 psi, 70 to 80 psi, 80
to 90 psi, 90 to 100 psi, 100 to 125 psi, 125 to 150 psi, 150 to
175 psi, 175 to 200 psi and overlapping ranges thereof. Additional
sources of shear (e.g., agitation, pumping, stirring etc.) are
optionally used in some embodiments to enhance the extraction of
PHA. Any one, or combination, of the PHA extraction methods
described herein, or disclosed in the art, may be utilized as a
method to carry out PHA extraction and remove PHA from the
PHA-containing biomass.
[0149] In several embodiments, a solvent-based extraction system is
utilized to carry out PHA extraction. In some embodiments, solvents
are utilized to carry out PHA extraction at high temperatures,
wherein PHA extraction occurs simultaneous with a
temperature-enhanced breakdown or dissolution of PHA-containing
biomass. In some embodiments, one or more solvent is utilized that
is biodegradable and metabolically assimilable by the culture, such
that PHA-reduced biomass comprising biomass and one or more
biodegradable solvent may be contacted with the culture, and both
the PHA-reduced biomass and the solvent may be utilized by the
culture as a source of carbon. Non-limiting examples of such
solvents include carbon dioxide, acetone, ethanol, methanol, and
methylene chloride, among others.
[0150] In several embodiments a mixture of solvent and PHA
comprises multiple phases, e.g. an aqueous phase and an organic
phase. In some embodiments, solvent-based extraction comprises a
more uniform mixture of solvent and PHA. In some embodiments,
depending on the solvent the phases are separated prior to recovery
of the PHA. In some embodiments, centrifugation is employed to
further distinguish and separate the phases of the mixture (e.g.,
separation of the solvent-PHA phase from the water-biomass phase).
In some embodiments, heat is also employed to maintain the
solubility of the PHA in a given solvent.
[0151] It shall be appreciated that the solubility of PHA varies
with the solvent used, and therefore the temperature (if adjusted)
and the separation techniques are tailored to match the
characteristics of a given solvent. Thus, in some embodiments
employing centrifugation, for example, a low speed centrifugation
is used to separate the solvent-PHA phase from the water-biomass
phase. In other embodiments, depending on the solvent, higher speed
centrifugation is used. In some embodiments, centrifugation is
employed in stages, e.g., low speed centrifugation followed by high
speed centrifugation. Any of a variety of centrifuges can be
employed, depending on the solvent used, for example, basket
centrifuges, swinging bucket centrifuges, fixed rotor centrifuges,
disc-back centrifuges, supercentrifuges, or ultracentrifuges.
[0152] In some embodiments, adjustable discharge ports suitable for
a particular centrifuge are used in order to control the rate and
degree of separation of solvent-PHA phase from the water-biomass
phase. In some embodiments, the concentration of water in the
water-biomass phase is adjusted to allow for suitable flow of the
mixture through the centrifuge (or within a centrifuge tube). For
example, in some embodiments, flow is suitable for separating the
phases when the concentration of biomass (relative to water) is
between about 1 and 100 g/L. In some embodiments, the concentration
is between about 10 to 20 g/L, 20 to 30 g/L, 30 to 40 g/L, 40 to 50
g/L, 50 to 60 g/L, 60 to 70 g/L, 70 to 80 g/L, 80 to 90 g/L, 90 to
100 g/L, 100 to 200 g/L, 200 to 400 g/L, 400 to 600 g/L, and
overlapping ranges thereof.
[0153] In still additional embodiments, increases in temperature
not only facilitate the extraction of the PHA, they also facilitate
the isolation of the PHA from the solvent (e.g. increased
temperature increases solvent evaporation).
[0154] In some embodiments, an extraction process is carried out to
remove PHA from a microorganism in such a manner that the
microorganism is deactivated. In some embodiments, the deactivation
is permanent, while in some embodiments the deactivation is
temporary. Without being bound by theory, it is believed that PHA
extraction techniques which do not permanently disable
microorganisms enable the PHA-reduced biomass generated thereby to
contribute to the metabolism of carbon sources after a PHA
extraction process, including through intracellular and
extracellular metabolism. In one embodiment, methods useful for the
temporary disablement of microorganisms include solvent extraction,
including solvent extraction carried out below 100.degree. C., and
particularly at intracellular temperatures below 100.degree. C.,
including extraction temperatures of about 10.degree. C. to
30.degree. C. to 50.degree. C. to 60.degree. C., 60.degree. C. to
70.degree. C., 70.degree. C. to 80.degree. C., 80.degree. C. to
90.degree. C., 90.degree. C. to 100.degree. C., and overlapping
ranges thereof.
[0155] In several embodiments, the PHA concentration of
PHA-containing biomass is reduced as a result of the PHA extraction
process. In several embodiments, the PHA concentration of
PHA-containing biomass is reduced by at least 0.01% (by dry cell
weight). In some embodiments, the PHA concentration is reduced by
about 10%-50%, 50% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85%,
to 90%, 90% to 95%, 95%-99.9%, and overlapping ranges thereof.
[0156] While a variety of methods are known to enable the
extraction PHA from biomass, most methods can be categorized into
one of two classes: a) solvent-based extraction, or b) NPCM
(non-polymer cellular material) dissolution-based extraction. NPCM
dissolution-based extraction methods utilize chemicals (such as
hypochlorite, or bleach), enzymes (such as protease), heat
(especially to reach temperatures above 100.degree. C.), pH (acids
and bases), and/or mechanical means (such as homogenization) to
break down, oxidize, and/or emulsify non-PHA cellular material. In
some cases, extraction methods from both categories can be
combined, such as the simultaneous utilization of hypochlorite and
methylene chloride.
[0157] NPCM dissolution-based extraction methods require continuous
and non-recoverable chemical inputs, such as hypochlorite,
peroxides, enzymes, and pH adjustors, and also generate significant
waste disposal issues. Thus, while these methods are effective, the
use of solvent-based extraction methods is generally preferred in
the industry due to the capacity of solvents to be distilled and
recovered for continuous re-utilization in a closed loop cycle.
Unfortunately, despite its benefits, some solvent-based extraction
methods are energy intensive processes that play a major role in
the high cost of PHA production, often accounting for more than 50%
of total production costs. Accordingly, there exists a significant
need for a novel method to significant increase the energy
efficiency of solvent-based extraction.
[0158] In several embodiments, a process for the extraction of
polyhydroxyalkanoates from biomass using a solvent-based extraction
method is provided, wherein the energy required to carry out the
process is reduced relative to prior solvent-based extraction
methods. Specifically, in one embodiment a high efficiency PHA
extraction process is provided comprising providing a
PHA-containing biomass comprising PHA and water, mixing the biomass
with a solvent at a temperature sufficient to dissolve at least a
portion of the PHA into the solvent and at a pressure sufficient to
enable substantially all or part of the solvent to remain in liquid
phase, thereby producing a PHA-lean biomass phase and a PHA-rich
solvent phase comprising solvent, water, and PHA, separating the
PHA-rich solvent phase from the PHA-lean biomass phase at a
temperature and pressure sufficient to enable substantially all or
part of the solvent to remain in the liquid phase and prevent
substantially all or part of the PHA within the PHA-rich solvent
phase from precipitating, reducing the pressure or increasing the
temperature of the PHA-rich solvent phase to cause the solvent to
vaporize and the PHA to precipitate or become a solid while
maintaining the temperature and/or the pressure of the PHA-rich
solvent phase to prevent all or part of the temperature-dependent
precipitation of the PHA into water, and collecting the solid PHA
material, including optionally separating the precipitated PHA from
the solvent and/or the water.
[0159] In the past, PHA precipitation has been induced in PHA-rich
solvent by a) adding a non-PHA solvent to the solvent phase to
reduce the solubility of PHA in the solvent phase and/or b)
reducing the temperature of the solvent phase to reduce the
solubility of PHA in the solvent. In particular, some methods 1)
dissolve PHA in a solvent by increasing the temperature of the
solvent and 2) precipitate PHA by reducing the temperature (and,
thus, solubility) of the solvent. Other methods require adding
water to a solution of PHA-rich solvent comprising dissolved PHA,
wherein the addition of water to the solution reduces the
solubility of the PHA in the solvent and causes the PHA to
precipitate into the solvent and/or water. (For example, U.S. Pat.
No. 4,562,245; U.S. Pat. No. 4,968,611; U.S. Pat. No. 5,894,062;
U.S. Pat. No. 4,101,533, all herein incorporated by reference.) In
each of these cases, energy efficiency is compromised;
specifically, by adding water or a non-PHA solvent to reduce the
PHA solubility of a solvent, additional energy is required for
downstream water/non-solvent removal, heating, and/or distillation.
By reducing the temperature of the solvent to reduce the solubility
of the solvent and induce PHA precipitation, heat energy is
redundantly expended, as the solvent must be re-heated for
distillation and recovery. Therefore, in several embodiments,
rather than adding a non-solvent to a PHA-solvent or reducing the
temperature of the PHA-solvent to effect PHA precipitation,
pressure and/or an increase in temperature is used to induce the
precipitation or solidification of the PHA without redundantly
reducing the temperature of solvent. Thus, in such embodiments,
there is a significant reduction in the energy required to heat
and/or distill non-solvent and/or solvent in downstream PHA
processing.
[0160] The removal of non-PHA materials from PHA often accounts for
a significant fraction of PHA production costs. As a specific
example, pigments often cause unwanted discoloration of PHA, and
must be removed through costly processes, such as ozonation,
peroxide washing, acetone washing, ethanol washing, solvent
refluxing, hypochlorite digestion, enzymatic degradation,
surfactant dissolution, or other methods disclosed in the art. In
several embodiments, a non-sterile process is used to select for
microorganisms exhibiting minimal pigmentation. Applicant has
surprisingly discovered that, by manipulating the concentration of
dissolved oxygen in a microorganism system, a culture may be
selected wherein white, tan, off-white, light brown, and/or light
yellow pigments are exhibited rather than purple, red, pink, dark
brown, orange, or other heavy pigments. Specifically, in some
embodiments, an excess of dissolved oxygen is introduced into a
growth media over successive periods, resulting in selective growth
of strains of methanotrophic microorganisms which produce white,
tan, off-white, light brown, and light yellow pigments, rather than
those producing pink, red, purple, dark yellow, dark brown, and/or
other heavy pigments. As a result, such embodiments, reduce the
need for costly downstream pigmentation removal.
[0161] As used herein, the term "PHA-reduced biomass" or
"substantially PHA-reduced biomass" shall be given its ordinary
meaning and shall be used to describe a biomass material wherein
the concentration of PHA relative to non-PHA material has been
reduced in a PHA-containing biomass through the utilization of a
PHA extraction process. In some embodiments, PHA-reduced biomass is
further treated in a variety of ways. In some embodiments, the
further treatment includes, but is not limited to, one or more of
dewatering, chemical treatment, sonication, additional PHA
extraction, homogenization, heat treatment, pH treatment,
hypochlorite treatment, microwave treatment, microbiological
treatment, including both aerobic and anaerobic digestion, solvent
treatment, water washing, solvent washing, and/or drying, including
simple or fractional distillation, spray drying, freeze drying,
rotary drying, and/or oven drying.
[0162] In one embodiment, PHA-reduced biomass is substantially
dried, wherein the resulting dried material comprises less than
about 99% liquids, including water, solvents, salts, and/or
growth-culture media. In some embodiments, the drying processes
disclosed herein yield a dried material containing between about
95% and 75% liquids, between about 75% and 50% liquids, between
about 50% and 25% liquids, between about 25% and 15% liquids
between about 15% and 10% liquids, between about 10% and 1%
liquids, and overlapping ranges thereof. In some embodiments,
drying is complete (e.g., between 1% 0.1% liquids, or less). In
another embodiment of the invention, a liquid phase comprising
PHA-reduced biomass is subjected to filtration, centrifugation,
density differentiation, or other method to increase the solids
content of the PHA-reduced biomass.
[0163] Traditionally, the separation of biomass from liquid growth
media is difficult and impractical due to the plugging and fouling
characteristics of biomass. In several embodiments, a method
enabling the efficient filtration of microorganisms is provided. In
some embodiments, a liquid chemical is added to the growth media
comprising microorganisms, wherein the liquid chemical is ethanol,
acetone, methanol, methylene chloride, ketones, alcohols, and/or
chlorinated solvents, or a combination thereof. In some
embodiments, microorganisms are then efficiently separated from
liquid growth media using standard filtration equipment, such as a
Buchner filter, filter press, or similar apparatus. In one
embodiment, approximately 2 parts acetone are mixed with one part
water, including both intracellular and extracellular water, to
effect the efficient filtration of microorganisms comprising the
water.
[0164] As used herein, the terms "isolated PHA" and "substantially
isolated PHA" shall be given their ordinary meaning and shall refer
to PHA that has been removed from a biomass material as a result of
an extraction process, or a biomass material wherein the
concentration of PHA relative to non-PHA material has been
increased by an extraction process. In several embodiments,
isolated PHA is further treated in one or more of a variety of
ways, including, but not limited to, purification, filtration,
washing, oxidation, odor removal, pigment removal, lipid removal,
non-PHA material removal, and/or drying, including centrifugation,
filtration, spray drying, freeze drying, simple or fractional
distillation, or density differentiation. Methods for the
purification of PHA include the use of peroxides, water,
hypochlorite, solvents, ketones, alcohols, and various other agents
to separate and remove non-PHA material from PHA material. In some
embodiments, PHA is removed from a microorganism culture by solvent
extraction to produce isolated PHA in a PHA-rich solvent phase and
PHA-reduced biomass in a PHA-lean liquid phase. In some
embodiments, the solvent phase is separated from the liquid phase
by filtration or centrifugation. In some embodiments, both
centrifugation and filtration are used in combination (e.g.,
sequentially). In some embodiments, centrifugation is optionally
followed by filtration. In other embodiments, filtration is
optionally followed by centrifugation. Filtration, in some
embodiments is performed under vacuum pressure, via gravity feed,
under positive pressure, or in specialized filtration centrifuges.
In some embodiments, the filter pore size is adjusted based on the
species composition of the microorganism culture. In some
embodiments, pore sizes of up to 200 .mu.m are used. In some
embodiments, smaller pore sizes are used, for example 15 to 20
.mu.m, 10 to 15 .mu.m, 5 to 10 .mu.m, 1 to 5 .mu.m, 0.001 to 1
.mu.m, and overlapping ranges thereof.
[0165] In addition to the steps outlined above, additional steps
may be taken to remove solvent from the extracted PHA, including
evaporation, solvent casting, steam stripping, heat treatment, and
vacuum treatment, each of which may be preferential,
cost-effective, time-effective, or advantageous depending on the
volatility and type of solvent used. In other embodiments, active
processes can be used to reduce the solvent content of the
solvent-PHA mixture. For example, in certain embodiments,
alterations in temperature of certain solvents change the
solubility of the PHA in the solvent, which effectively removes
solvent from the PHA (e.g., the solvent is now separable from a
precipitated PHA). In some embodiments, filtration, solvent
temperatures, or vacuum treatment can be increased to reduce a
portion of the solvent. In some embodiments, solvent to PHA ratios
post extraction, filtration, evaporation, solvent casting, steam
stripping, heat treatment, and/or vacuum treatment range from about
0.1:1 to about 1,000:1, including about 0.2:1, 0.3:1, 4.0:1, 5.0:1,
10.0:1, 20.0:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 500:1, and
900:1.
[0166] As a result of the processes disclosed above, in some
embodiments, the solvent is substantially removed from the isolated
PHA in the PHA-rich solvent phase and the liquid is substantially
removed from the PHA-reduced biomass in the PHA-lean liquid phase.
In some embodiments, the isolated PHA is dried in a heated vessel
to produce substantially pure isolated PHA (e.g., at least 80% PHA
by dry weight, preferably at least 98% PHA by weight, more
preferably at least 99% PHA by weight).
[0167] Numerous varieties of heated or drying vessels may be used
to dry the isolated PHA, including ovens, centrifugal dryers, air
dryers, spray dryers, and freeze dryers, among others. In some
embodiments, heat is applied to a drying vessel to speed the
process and/or to remove (e.g., evaporate traces of solvent from
the PHA). It shall be appreciated that the moisture content of the
isolated PHA will depend, in some embodiments, on the solvent used,
and the corresponding separation technique used (as described
above). For example, a volatile solvent in combination with
ultracentrifugation would result in a less moist extracted PHA,
while a less active separation technique (e.g., gravity phase
separation) would yield a more moist extracted PHA. In some
embodiments, internal dryer temperatures range from 20.degree. C.
to 40.degree. C. to about 200.degree. C. In some embodiments,
internal temperatures range from about 50.degree. C. to 90.degree.
C., about 90.degree. C. to 180.degree. C., about 65.degree. C. to
175.degree. C. and overlapping ranges thereof. In some embodiments,
outlet temperatures are substantially lower than inlet on internal
temperatures. In some embodiments, outlet temperatures range from
30.degree. C. to 90.degree. C. In some embodiments, outlet
temperatures are between about 35.degree. C. to 40.degree. C.,
about 40.degree. C. to 45.degree. C., about 45.degree. C. to
50.degree. C., about 50.degree. C. to 55.degree. C., about
55.degree. C. to 90.degree. C., and overlapping ranges thereof. It
shall also be appreciated that the internal and outlet temperatures
may be adjusted throughout the drying process (e.g., the
temperature difference may initially be large, but decrease over
time, or vice versa).
[0168] From the above discussion, it shall be appreciated that the
type of dryer used, and the temperatures used (if other than
atmospheric temperatures) are easily tailored to correspond to the
techniques used in the extraction process. In some embodiments,
particular dryer components are beneficial in the isolation of PHA.
For example, depending on the moisture content of the extracted PHA
(e.g., the amount of residual solvent) particular components of an
evaporative-type dryer, such as an oven dryer, rotary dryer, spin
flash dryer, spray dryer (equipped with various types of nozzle
types, including rotary atomizor, single flow atomizer, mist
atomizer, pressure atomizer, dual-flow atomizer) convection heat
dryer, tray dryer, scrape-flash dryer, or other dryer type are
used. By way of additional example, if a freeze dryer (e.g., a
lyophilizer) is used, in some embodiments a manifold dryer is used,
optionally in conjunction with a heat source. Also by way of
example, a tray lyophilizer can be used, in some embodiments, with
the isolated and dried PHA being stored and sealed in containers
(e.g., vials) before re-exposure to the atmosphere. In certain
embodiments, such an approach is used when long-term storage of the
PHA is desired.
[0169] It shall also be appreciated that certain varieties of
heated/drying apparatuses have adjustable flow rates that can be
tailored to the moisture content of the isolated PHA. For example,
an isolated PHA having a high moisture content would be fed into a
dryer at a slower input rate to allow a higher degree of drying per
unit of PHA inputted into the dryer. Conversely, a low moisture
content isolated PHA would likely require less time to dry, and
therefore could be input at a faster rate. In some embodiments,
input rates of isolated PHA range from several hundred liters of
isolated PHA-solvent mixture per minute down to several milliliters
per minute. For example, input rates can range from about 10 mL/min
to about 6 L/min, including about 10 ml/min to about 50 ml/min,
about 50 mL/min to about 100 ml/min, about 100 ml/min to about 500
ml/min, about 500 ml/min to about 1 L/min, about 1 L/min to about 2
L/min, about 2 L/min to about 4 L/min, about 4 L/min to about 6
L/min, and about 100 L/min to about 500 L/min.
[0170] A range of PHA functional characteristics can be attained by
mixing one PHA molecule, such as PHB, with various PHA polymers,
including PHB, at various molecular weights. Therefore, in several
embodiments, a first isolated PHA is heated to reduce the molecular
weight of the first isolated PHA, and then subsequently mixed with
a second PHA wherein the molecular weight of the second PHA is
higher than the molecular weight of the first PHA. With such
embodiments, Applicant has surprisingly discovered methods to
functionalize one or more PHAs, including PHB. In additional
embodiments of the invention, the molecular weight of a first PHA
is reduced from about 800,000-5,000,000 Daltons to about 30,000 to
800,000 Daltons and mixed with a second PHA with a molecular weight
of about 800,000 to 5,000,000 Daltons to modify the functionalities
of the input PHAs. In yet another embodiment, a first PHA is mixed
with a second PHA wherein the molecular weight of the first PHA is
at least 0.1% less than the molecular weight of the second PHA. In
some embodiments, the difference in molecular weight between the
first and second PHA is about 0.1% to 1%, about 1% to 10%, about
10% to 20%, about 20% to 30%, about 30% to 40%, about 40% to 50%,
about 50% to 60%, about 60% to 70%, and overlapping ranges thereof.
In still additional embodiments, PHAs having greater differences in
molecular weight are used. In yet another embodiment, the molecular
weight of a first PHB is reduced to less than about 100,000-500,000
Daltons and mixed with a second PHA with a molecular weight greater
than about 100,000-500,000 Daltons to modify the functionality of
the input PHB. It shall be appreciated that input PHA and PHB
weight may vary from the ranges disclosed above, but based on the
differences in the molecular weights, the alteration in
functionality of the input PHB is still achieved.
Purifying the Isolated PHA
[0171] In some embodiments, isolated PHA is purified to produce a
PHA material that is substantially pure PHA. In some embodiments,
the isolated PHA is purified to at least 20% pure PHA by dry
weight. In some embodiments, the isolated PHA is purified to at
least 55% pure PHA by dry weight, while in some embodiments, the
isolated PHA is purified to at least 90% pure PHA by dry weight. In
additional embodiments, purity of the isolated PHA is between about
90 and 99.9%, including 91, 92, 93, 94, 95, 96, 97, 98, and 99%
pure.
[0172] In several embodiments, isolated PHA may be recovered by any
one, or a combination, of the methods described above, including,
but not limited to: washing, filtration, centrifugation,
dewatering, purification, oxidation, non-PHA material removal,
solvent removal, and/or drying. In some embodiments, isolated PHA
is recovered according to the manner in which it has been removed
from the culture. For example, in embodiments in which
solvent-based extraction is employed, a recovery method may be
employed to remove the isolated PHA from the solvent and/or other
non-PHA material. In one embodiment, solvent may be used to extract
the PHA, wherein the solvent is then substantially removed from the
isolated PHA by carrying out PHA precipitation and filtration,
excess solvent distillation and/or removal, and/or drying,
resulting in the recovery of dry, isolated PHA. In embodiments
employing enzyme, surfactant, protonic, hydroxide, and
hypochlorite-based extraction techniques wherein the dissolution of
non-PHA material is induced, isolated PHA may be filtered, washed,
separated, centrifuged, and/or dried, resulting in the recovery of
dry, isolated, purified PHA. The resultant PHA, in some
embodiments, is further used in downstream processing, including
thermoforming.
Returning PHA-Reduced Biomass to the PHA-Producing Culture to
Convert PHA-Reduced Biomass into PHA
[0173] In several embodiments of the invention, the PHA-reduced
biomass is returned to the culture to cause the biomass-utilizing
microorganisms within the culture to convert the carbon within the
PHA-reduced biomass into PHA. By using PHA-reduced biomass as a
source of carbon for the production of microorganisms in a
microorganism fermentation system, a series of biochemical
enzymatic pathways are generated in situ by the microorganism
culture to carry out the metabolic utilization of PHA-reduced
biomass for growth, reproduction, and PHA synthesis.
[0174] Without being limited by theory, it is believed that
gas-utilizing microorganisms and biomass-utilizing microorganisms
are able to co-exist as a single microorganism consortium because
they utilize sources of carbon that require distinctly different
bioenzymatic assimilation pathways. For instance, while methane
metabolism requires the methane monooxygenase enzyme to catalyze
the conversion of methane into methanol for cellular assimilation,
and methane monooxygenase is competitively inhibited by a wide
range of compounds, it is not inherently deactivated by high
concentrations of cellular biomass, including PHA-reduced biomass.
Similarly, the chlorophyll-based metabolic assimilation systems
required for the conversion of carbon dioxide into biomass and PHA
are not inherently deactivated or competitively inhibited by high
concentrations of cellular biomass, including PHA-reduced biomass.
Likewise, the enzymatic architecture enabling the metabolic
utilization, breakdown, and/or assimilation of PHA-reduced biomass
is not inherently deactivated or competitively inhibited by high
concentrations of methane and/or carbon dioxide, particularly as
the process requires neither methane monooxygenase nor chlorophyll.
Without being limited by theory, Applicant believes that the
relatively non-competitive, and in some cases commensal or
mutualistic relationships between microorganisms consuming a
carbon-containing gas and a PHA-reduced biomass, make it possible
to create a microorganism culture comprising biomass-utilizing
microorganisms and gas-utilizing microorganisms, wherein both
carbon-containing gases and PHA-reduced biomass may be metabolized
as simultaneously assimilable sources of carbon.
[0175] In the case of autotrophic, methanotrophic, and/or
biomass-utilizing microorganisms, Applicant has found that a
mutualistic, positive-feedback loop relationship can be created in
a single (or optionally multiple) reaction vessel. In such
embodiments, the oxygen created by autotrophic metabolism is
utilized by methanotrophic and/or biomass-utilizing microorganisms
for metabolic functions, the carbon dioxide created by
methanotrophic and/or biomass-utilizing microorganism metabolism is
utilized for autotrophic metabolism, the methane and/or carbon
dioxide created by anaerobic methanogenic microorganisms is
utilized by methanotrophic microorganisms, and the biomass created
by methanotrophic, autotrophic, and/or heterotrophic microorganisms
is used to provide a source of carbon to methanogenic and/or other
heterotrophic microorganisms. Due to the microscopic-level
induction of oxygen and/or carbon dioxide created therein, mass
transfer efficiencies in several embodiments are significantly
improved over traditional gas induction means, such as gas
sparging, mechanical mixing, static mixing, or other means known in
the art. To applicant's knowledge, prior to the disclosure herein,
the use of autotrophic microorganisms cultured in association with
heterotrophic microorganisms has never been suggested as a means to
improve mass transfer efficiencies, supply oxygen, and/or augment
microorganism growth rates in a positive feedback loop system.
[0176] In several further embodiments of the invention, PHA-reduced
biomass is used by heterotrophic microorganisms, including
acidogenic, acetogenic, and methanogenic microorganisms, to produce
methane, which is further utilized by methanotrophic microorganisms
to produce biomass, including PHA. In some embodiments of the
invention, anaerobic microorganisms coexist with aerobic
microorganisms under microaerobic conditions (e.g., mean dissolved
oxygen concentrations approximately 0.00-1.0 ppm, including about
0.001 to 0.002 ppm, 0.002 to 0.03 ppm, 0.03 to 0.04 ppm, 0.04 to
0.5 ppm, 0.5 to 0.6 ppm, 0.6 to 0.7 ppm, 0.7 to 0.8 ppm, 0.8 to 0.9
ppm, 0.9 to 1.0 ppm, and overlapping ranges thereof.
[0177] In some embodiments of the invention, heterotrophic,
methanotrophic, methanogenic, and/or autotrophic microorganisms are
divided into multiple stages and vessels, in particular, anaerobic
and aerobic stages, in order to carry out the conversion of
PHA-reduced biomass into methane and PHA. In further embodiments of
the invention, PHA-reduced biomass is returned to the culture using
one or more vessels, whereby it is first converted to carbon
dioxide, methane, and/or volatile organic compounds by a
heterotrophic, e.g., methanogenic, microorganism consortium under
anaerobic conditions and then converted to PHA by methanotrophic
microorganisms under aerobic conditions, whereby carbon dioxide is
also metabolized or otherwise used by autotrophic microorganisms,
methanotrophic microorganisms, and heterotrophic
microorganisms.
[0178] In several embodiments, light intensity is utilized to
regulate the growth rate of heterotrophic and/or methanotrophic
microorganisms. In some embodiments, light intensity is manipulated
to regulate the generation of oxygen by autotrophic microorganisms.
In some embodiments, the rate of oxygen generated by autotrophic
microorganisms is subsequently used to control the growth and
metabolism of heterotrophic and methanotrophic microorganisms.
[0179] In several embodiments, carbon dioxide is supplied to
autotrophic microorganisms in the form of carbon dioxide created by
methanotrophic and/or heterotrophic microorganisms. In some
embodiments, each of these varieties of microorganism is cultured
in a single vessel. In some embodiments, methane, sugar, biomass,
and/or another non-carbon dioxide source of carbon is used to grow
autotrophic microorganisms. To applicant's knowledge, autotrophic
microorganisms have never been cultured using methane as a sole
carbon input.
[0180] Some gas-utilizing microorganisms are unable to produce high
concentrations of intracellular PHA. However, according to several
embodiments, certain microorganism consortiums utilizing
PHA-reduced biomass, or derivatives thereof, as a source of carbon
are able to generate high intracellular PHA concentrations and thus
effectively convert low PHA concentration biomass derived from a
carbon-containing gas into a high PHA concentration biomass
material under the conditions disclosed herein. In several
embodiments, the concentration (by weight) of intracellular PHA is
between about 10% to 30%, 30% to 50%, 50% to 70%, 70% to 80%, 80%
to 90%, 90% or more, and overlapping ranges thereof. Thus, in one
embodiment, the culture is contacted with the PHA-reduced biomass
and then manipulated, according to the processes described herein,
to effect PHA synthesis, wherein the PHA-reduced biomass is
converted into PHA by biomass-utilizing microorganisms. In some
embodiments, PHA synthesis is induced by nutrient limitation,
nutrient excess, nutrient imbalance, or large shifts in nutrient
concentration. In still further embodiments, PHA synthesis is
induced by reducing the availability of nitrogen, oxygen,
phosphorus, or magnesium to the culture. In some embodiments, these
nutrients are simultaneously reduced (to varying or similar
degrees). In some embodiments, the nutrients are reduced
sequentially. In some embodiments, only one of the nutrients is
reduced. For example, in certain embodiments, PHA synthesis is
induced by reducing the availability of oxygen to the culture. In
some embodiments, this is achieved by manipulating the flow rate of
air or oxygen into the growth medium. In some embodiments,
manipulation of the flow rate of other carbon-containing gases,
such as methane and/or carbon dioxide, into the growth medium, or
otherwise manipulating the rate of gas transfer in a system (e.g.,
by adjusting mixing rates or light injection rates) is employed. In
one embodiment, oxygen limitation is induced by reducing the flow
rate of oxygen into the growth medium. In another embodiment,
oxygen limitation is induced by reducing the rate of light
transmission into the medium to reduce the production of oxygen by
autotrophic microorganisms. In some embodiments of the invention,
the concentration of PHA generated in a biomass-utilizing
microorganism culture utilizing PHA-reduced biomass as a source of
carbon is at least 5%, at least 20%, or at least 50% by dry cell
weight; in particularly preferred embodiments of the invention, the
concentration of PHA in a biomass-utilizing microorganism is at
least 80% by dry cell weight.
[0181] In some embodiments, a PHA-reduced biomass recycling system
is utilized wherein substantially all (e.g., at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, or at least 98%) of
the PHA-reduced biomass produced is contacted with the culture
until it is converted into PHA. In some such embodiments, solid
sources of carbon are substantially output from the process or
culture only in the form of isolated PHA.
[0182] In several embodiments, as carbon-containing gases are
continually added to the medium to effect the production of
biomass, the process disclosed above is repeated. Specifically, as
the process continues, a portion of the PHA-containing biomass from
the culture is removed from the medium, PHA is extracted from the
PHA-containing biomass, PHA-reduced biomass is separated from
isolated PHA, isolated PHA is recovered, purified, and dried, and
PHA-reduced biomass is sent back to the culture and converted by
the culture into PHA. In one embodiment, substantially all
PHA-reduced biomass produced is contacted with the culture until it
is converted into isolated PHA, and solid sources of carbon are
substantially output from the process only in the form of isolated
PHA. In other embodiments, carbon is substantially output from the
system only in the form of PHA and methane, carbon dioxide, and/or
volatile organic compounds.
[0183] The following example is provided to further illustrate
certain embodiments within the scope of the invention. The example
is not to be construed as a limitation of any embodiments, since
numerous modifications and variations are possible without
departing from the spirit and scope of the invention.
Example 1
[0184] A fermentation system comprising one or more vessels are
partially filled with one or more liquid growth mediums, wherein
the medium comprises methanotrophic, autotrophic, methanotrophic,
and/or other heterotrophic or biomass-utilizing microorganisms
containing PHA, and, per liter of water, 0.7-1.5 g
KH.sub.2PO.sub.4, 0.7-1.5 g K.sub.2HPO.sub.4, 0.7-1.5 g KNO.sub.3,
0.7-1.5 g NaCl, 0.1-0.3 g MgSO.sub.4, 24-28 mg
CaCl.sub.2*2H.sub.2O, 5.0-5.4 mg EDTA Na.sub.4(H.sub.2O).sub.2,
1.3-1.7 mg FeCl.sub.2*4H.sub.2O, 0.10-0.14 mg CoCl.sub.2*6H.sub.2O,
0.08-1.12 mg MnCl.sub.2*2H.sub.2O, 0.06-0.08 mg ZnCl.sub.2,
0.05-0.07 mg H.sub.3BO.sub.3, 0.023-0.027 mg NiCl.sub.2*6H.sub.2O,
0.023-0.027 mg NaMoO.sub.4*2H.sub.2O, 0.011-0.019 mg
CuCl.sub.2*2H.sub.2O. One or more of the mediums are anaerobic
and/or aerobic, and carbon containing gases, including methane,
carbon dioxide, and volatile organic compounds, as well as
optionally air or oxygen, are fed into all or part of the system to
induce the growth and reproduction of microorganisms through the
utilization of carbon-containing gases, as well as the production
of PHA.
[0185] Next, a portion of the media volume of the fermentation
system is passed through a basket centrifuge to increase the solids
content of the medium to approximately 167 g/L. The
solids-containing centrate phase of the centrifuged solution is
then transferred to a PHA extraction vessel, and the substantially
solids-free filtrate phase of the centrifuged solution is recycled
back to the fermentation system.
[0186] In some embodiments, the solids-containing centrate phase is
optionally chemically pre-treated prior to extraction. In some
embodiments, one or more of acids, bases, chloride, ozone, and
hydrogen peroxide is added. In several embodiments, chemical
pre-treatment increases the efficiency and yield of the subsequent
extraction process. In some embodiments, the chemical pre-treatment
functions to break down the cell well (partially or fully), thereby
liberating a greater portion of the intracellular PHA. In some
embodiments, chemical pre-treatment dissolves and/or removes
impurities that negatively impact the PHA extraction process. In
some embodiments, chemical pre-treatment enhances cell
agglomeration, which increases the percentage of microorganisms
that are extracted (e.g., cells in an agglomerated mass are not
separated or lost in transfer steps). Next, following optional
chemical pre-treatment, solvent is added into the PHA extraction
vessel to create a solvent solution, and the solvent solution is
then mixed for a period of time to cause the PHA in both the
microorganisms to dissolve into the solvent, and thereby create
PHA-rich solvent and PHA-reduced biomass. Over the course of a
defined mixing period (e.g., 0.1-10 hours), the PHA content of the
microorganisms is reduced by about 80% as it is dissolved into the
solvent.
[0187] Next, the solvent solution comprising the PHA-rich solvent
and PHA-reduced biomass is passed through a filter located at the
bottom of the PHA extraction vessel, and the PHA-rich solvent is
thereby separated from the PHA-reduced biomass. Water is then added
to the PHA extraction vessel to create a water-biomass solution,
and the water-biomass solution is then heated to 75.degree. C. to
cause any remaining solvent associated with the PHA-reduced biomass
to exit the PHA extraction vessel as a gaseous vapor. The vapor
discharged from the PHA extraction vessel is then passed through a
heat exchanger and recovered as liquid solvent. Meanwhile, the
PHA-rich solvent is transferred to a PHA purification vessel and
mixed with room temperature water to create a water-solvent
solution. The water-solvent solution is then heated to cause i) the
solvent to exit the PHA extraction vessel as a gaseous vapor and
ii) the isolated PHA to precipitate into the water and/or become a
solid. The solvent vapor created by heating the water-solvent
solution is then passed through a heat exchanger and converted into
liquid solvent.
[0188] The isolated PHA is then substantially dewatered by
filtration in a Nutsche filter, and the Nutsche filter containing
the substantially dewatered isolated PHA is then heated to remove
any additional volatile compounds, including trace water and/or
solvent. Following heat drying in the Nutsche filter, the isolated
PHA is recovered as substantially pure PHA (e.g., greater than
about 90% PHA).
[0189] Concurrently, the water-biomass solution comprising
PHA-reduced biomass and water is transferred from the PHA
extraction vessel back into the fermentation system, where the
PHA-reduced biomass is contacted with one or more of the
microorganism cultures. Next, the medium of the fermentation system
is manipulated to cause the one or more microorganisms within the
system to metabolize the PHA-reduced biomass as a source of carbon
and nutrients.
[0190] Depending on the embodiment, the culture conditions are
adjusted to determine the point at which the inception of the
growth or PHA metabolism phase occurs. As discussed herein,
manipulation of the concentration of one or more growth culture
media nutrients can alter the metabolic pathways favored by certain
microorganisms. Additionally, the use of PHA-reduced
biomass-derived carbon for the production of additional biomass
versus the production of PHA can be tailored based on whether the
intent is to grow the culture (e.g., increase the overall biomass)
or to harvest PHA (e.g., shift the culture from growth phase to
production of PHA). As such, the reduction, increase, or adjustment
of the concentration certain growth nutrients, and the timing of
such adjustment, plays a role in the metabolic state and PHA
production of the culture. Adjustment of growth nutrients can occur
at any point after the PHA-reduced biomass is returned to the
microorganism system. In some embodiments, adjustment is immediate
(e.g., within minutes to a few hours). In some embodiments, a
longer period of time elapses. In some embodiments, adjustment in
one or more growth nutrients occurs after about 2 to 4 hours, after
about 4 to 6 hours, after about 6 to 8 hours, after about 8 to 10
hours, after about 10 to 12 hours, after about 12 to 14 hours,
after about 14 to 18 hours, after about 18 to 24 hours, and
overlapping ranges thereof. In still additional embodiments,
adjustment in one or more growth nutrients occurs after about 2 to
5 days, 5 to 10 days, 10 to 15 days, 15 to 20 days, 20 to 30 days,
30 to 50 days, and overlapping ranges thereof. In some embodiments,
longer times elapse prior to adjusting one or more growth nutrients
to induce PHA polymerization.
[0191] After a desired period of time has elapsed, the dissolved
oxygen and/or nitrogen concentration (or concentration of another
nutrient) of one or more parts of the medium is reduced or adjusted
to cause one or more of the microorganisms within the system to
utilize the PHA-reduced biomass in the medium as a source of carbon
for the synthesis of PHA. In some embodiments, the percent
adjustment ranges from about 20% to about 100%, including 20% to
30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%,
80% to 90%, 90% to 100%, and overlapping ranges thereof. It shall
be appreciated that, depending on the characteristics of a culture
in a given embodiment, a specific percentage reduction, increase,
or adjustment in nutrient may not be necessary, but a reduction,
increase, or adjustment is used that is sufficient to convert
certain cells from a relative growth phase to a relative PHA
synthesis phase. After approximately 12-24 hours of PHA synthesis,
substantially all of the PHA-reduced biomass within the growth
medium has been metabolized into biomass-utilizing microorganism
biomass, including PHA. It shall be appreciated that, in certain
embodiments, greater or lesser PHA synthesis times result in
varying percentages of the PHA-reduced biomass within the growth
medium being metabolized into biomass, including PHA.
[0192] As carbon containing gases are continually added to the
fermentation system to effect the production of biomass, the
process is repeated, wherein solid sources of carbon substantially
exit the system only in the form of PHA. Specifically, as the
process continues, a portion of the PHA-containing biomass from the
fermentation vessel is passed through a dewatering centrifuge to
increase the solids content of the PHA-containing biomass, PHA is
extracted from the removed PHA-containing biomass using a
solvent-based extraction system to create PHA-reduced biomass and
isolated PHA, PHA-reduced biomass is separated from isolated PHA,
isolated PHA is recovered, purified, and dried, and PHA-reduced
biomass is sent back to the fermentation system and converted by
microorganisms into PHA, such that substantially all PHA-reduced
biomass produced is contacted with the culture until it is
converted into isolated PHA, and wherein solid sources of carbon
are substantially output from the process only in the form of
isolated PHA.
[0193] While the above description of several compositions,
systems, and methods contains many specificities, it should be
understood that the embodiments of the present invention described
above are illustrative only and are not intended to limit the scope
of the invention. Numerous and various modifications can be made
without departing from the spirit of the embodiments described
herein. Accordingly, the scope of the invention should not be
solely determined by the embodiments described herein, but also by
the appended claims and their legal equivalents.
* * * * *